International travellers will have to provide a negative pre-departure test taken two days before travelling, the Scottish Government announced at 7pm.
They will also need a negative PCR test on or before day two after arrival, under measures agreed on a UK four-nation basis, a spokesperson said.
In addition, Nigeria has been added to the red list for international travel from 4am on Monday, with all travellers returning to Scotland from Nigeria required to quarantine in managed accommodation for ten days.
It will join ten African countries on the red list: Angola, Botswana, Eswatini, Lesotho, Malawi, Mozambique, Namibia, South Africa, Zambia and Zimbabwe.
Transport Secretary Michael Matheson said: “It is essential we take steps now to keep people safe, protect the roll out of the booster programme and reduce the chances of unsustainable pressure being placed on the NHS over the winter.
“We have always said it may be necessary to quickly implement fresh measures to protect public health in Scotland, particularly with regards to international travel, and these restrictions are proportionate and necessary to that aim.
“We fully understand the impact the changes will have on staff and businesses in the travel and aviation sectors, particularly as the new variant came at a time when we were beginning to see some signs of recovery. We will not keep the restrictions in place any longer than is necessary.”
UK Transport Secretary Grant Shapps tweeted: “As the scientists work to understand new Omicron variant, we need to apply additional caution until picture is clearer.
"We appreciate this will be difficult for the travel sector as we prioritise public health and protect the progress of our world-leading vax & booster programme.”
The latest tightening of travel restrictions follows pressure from Labour and scientists following the introduction this week of PCR tests, but only within two days of arrival.
Shadow home secretary Yvette Cooper welcomed the move but said ministers should have moved sooner.
She said: "We badly need them to learn the lessons on the importance of acting quickly on Covid border measures rather than each time having to be put under huge pressure to finally act."
The BBC reported that the UK Government’s Scientific Advisory Group for Emergencies (Sage) decided on Monday that pre-departure testing would be "valuable".
The UK Health and Security Agency (HSA) said today a further 26 cases of the Omicron variant have been reported across the UK - 25 of them in England.
It takes the total number of confirmed cases of the variant in the UK to 160, including 30 in Scotland.
A risk assessment by the HSA rated the Omicron variant as "red" for severity of infection and "amber" for transmissibility between humans.
It said the variant was likely to reduce the protection from both naturally or vaccine-acquired immunity.
However it acknowledged there was so far "insufficient data" to reach firm conclusions and the assessment was presented with "low confidence".
HSA chief executive Dr Jenny Harries said: "We are working as fast as possible to gather more evidence about any impact the new variant may have on severity of disease or vaccine effectiveness.
"Until we have this evidence, we must exercise the highest level of caution in drawing conclusions about any significant risks to people's health.”
The Scottish Passenger Agents’ Association (SPAA), which said it represented “truly battle weary” travel agents and firms, described the new tests as a “crushing blow”.
President Joanne Dooey said: “The confidence which was returning to international travel will be stamped out following this announcement.
“It leaves everyone currently overseas who has a scheduled return to the UK from 4am this Tuesday scrambling to find testing which meets the Scottish Government regulations.
“We expect that travel agents’ phones will be ringing off the hook with customers who wish to cancel or postpone their holidays, business trips and Christmas visits to families overseas.
"The reintroduction of pre-departure testing and the fact that countries can be placed on the ‘red list’ with less than 48 hours’ notice will plunge agents back into a chaotic world of client repatriations.
"We’re asking that the Scottish Government recognises our members need sector-specific support urgently.”
The Airport Operators Association said it would have a “devastating” impact on tourism and aviation.
Chief executive Karen Dee said: “Pre-departure tests acts as a major deterrent to travel and most of the limited remaining demand following the reintroduction of self-isolation will now fall away, just as airports were hoping for a small uplift over the Christmas holiday.
“Travel and aviation are the only sectors hit with any operating restrictions in response to the Omicron variant.”
Rory Boland, Which? Travel Editor, said: "Travellers will understand the need for government to introduce public health measures, however constantly changing rules at the last minute leaves passengers footing the bill.
"In some destinations, it will be difficult for people to secure tests at such short notice.
"For those who do test positive abroad, they should first and foremost follow local health guidelines.
"This will likely require quarantining in your existing hotel or at government accommodation.
"In some countries, travellers will have to pay for quarantine, and some travel insurance providers can help with this.
"Travellers should also check the flexible booking policy of their airline to rearrange flights."
British Airways chairman and chief executive Sean Doyle said: "The blanket re-introduction of testing to enter the UK, on top of the current regime of isolation and PCR testing on arrival, is completely out of step with the rest of the world, with every other country taking a measured approach based on the science.
"Our customers will now be faced with uncertainty and chaos and yet again this a devastating blow for everyone who works in the travel industry."