Organisers of the Missing EU Already event have promised "speakers, a music light show, a time for reflection, plus anthem singing" when it takes place on Friday at 6pm.
Among those scheduled to speak outside Holyrood is Lorna Slater, co-convener of the Scottish Greens, and David Clarke, vice-chairman of the European Movement in Scotland.
The event is being co-organised by several pro-EU groups in the Capital, with similar gatherings planned in other towns across Scotland.
The EU has said an extension to the negotiations can be agreed by the end of June - but Boris Johnson has repeatedly said that any deal must be agreed by the end of the year.
Mark Lazarowicz, chairman of the European Movement in Scotland, said: “Leaving the European Union is an incredibly sad event and this series of activities provides an opportunity to not only celebrate our membership and the great benefits it has given us, but also to look to the future.
“While we may have left, the fight now begins for us to rejoin the EU, and that fight starts now.”
The UK Government is planning a series of low-key events to mark Brexit Day. The front of 10 Downing Street will be turned into a digital Brexit countdown clock, which will start ticking down from 10pm.
Union flags will line The Mall and Parliament Square with government buildings lit up in red, white and blue in a light show designed to "symbolise the strength and unity of our four nations".
Calls by several Tory MPs for Big Ben - which is currently undergoing long-term restoration work - to chime to mark Brexit Day were overruled on cost grounds.
Nigel Farage is backing a “Brexit Celebration” event hosted by Leave Means Leave in Parliament Square which is expected to attract thousands of supporters and will feature speeches by senior Brexiteers.