They are appealing for support to save the service from privatisation under the latest phase of proposed NHS reforms.
Since 2004, the service has been provided by Great Western Ambulance Service for NHS Gloucestershire after local GPs gave up out of hours work. Bidders will be able to tender for a new contract to run the service from September 2013.
The move could see the service awarded to profit making businesses like Virgin or Harmoni.
Tanya Palmer, regional manager in UNISON South West, said: "After over eight years of running a highly successful service GWAS, and the public of Gloucester, are faced with an uncertain future. The current visiting service is not failing.
"Private companies are rapidly swallowing up local services and it's important we do all we can to keep this, and other, excellent services within the NHS and with local GPs."
In March, a new 111 service will replace the existing NHS Direct. Campaigners are concerned introducing two new untested services within a few months could impact on patient care.
Shane Clark, representative of Great Western Ambulance branch of UNISON, said: "Very little will be known about this new service and how it will impact on the out of hours services and other NHS services for a least two years. Putting the out of hours services out to tender at the same time could prove disastrous for the health and wellbeing of those in Gloucestershire."
Clinical Chair of Gloucestershire Clinical Commissioning Group, Dr Helen Miller said:
"The CCG will take on its formal responsibilities from April 1,2013 and is led by experienced clinicians who are passionate about securing the highest quality NHS services for local people.
"Our constitution will be extremely strong and clear in the areas of accountability."
The petition can be found at http://you.38degrees.org.uk/petitions/keep-gloucestershire-out-of-hours-ooh-within-the-nhs.