Umbrella Lane will relaunch the fund on Saturday to ensure sex workers and their families have "some financial support in these challenging times".
Charity founder Anastacia Ryan said: "Sex worker-led charities have repeated asked the Government for crisis support.
"The Government response has been that there was already a 'bespoke fund' created for women in prostitution. This fund was only given to mainstream organisations that fail to work from a rights-based, trauma-informed approach, which is part of the reason the funds have not reached those hardest hit.
"Due to stigma and often criminalisation, sex workers are untrusting of these services, which is why it is essential that crisis funds are made available to sex worker-led charities to administer to those who need it most."
Claire, a sex worker in Glasgow, said when Umbrella Lane's initial hardship fund ran out, she "felt forced to choose between seeing clients and risking my health and safety or facing the prospect of losing my home and not being able to put enough food on the table for my young kids".
Prerna Menon, Umbrella Lane co-ordinator said: "The hardship fund will be a vital resource available to all sex workers in Scotland who are having problems meeting their basic needs in order to offer them a small respite from the extreme hardship they are experiencing."
A Scottish Government spokeswoman said: "We know that these challenging and unprecedented times are impacting those involved in prostitution.
"Social Security such as Universal Credit and Housing Benefit is available to those eligible and other financial support, such as the Scottish Welfare Fund, is also available to those on low incomes.
"We've allocated more than £60,000 crisis funding to the Encompass Network to enable Covid-focused services for destitution support, trauma counselling, advocacy and to provide a bridge to national mainstream support.
"This fund remains open and is available to women who have been involved in selling or exchanging sex and are affected by the pandemic with no alternative options - this fund can be accessed privately and is not conditional on exiting prostitution."
Reporting by PA