The petition, addressed to concert organiser Mediacorp, the Singapore government and culture minister Grace Fu, says a performance by the openly gay Lambert is inappropriate and does not align with Singaporean values.
The concert organiser said it was sticking with Lambert, a runner-up on American idol who caused controversy during his2009 American Music Awards performance when he kissed his male keyboardist and stimulated sexual acts with dancers.
Sex between men is illegal in Singapore.
"We urge the organisers of Countdown 2016 to recognise and respect the values of the majority of Singapore that has voiced its desire to preserve our nation's moral fibre," reads the petition, which was posted anonymously by a group saying it represented concerned parents. (http://bit.ly/1lNoM5N)
The petitioners argue that having Lambert at the concert would bring a sour note to the end of the conservative city-state's 50th birthday celebrations.
State TV company Mediacorp maintained Lambert would perform, and said the concert would be suitable for family audiences and conform with broadcast regulations.
Not all of Singapore wants to exclude the singer. A rival online petition has so far gathered 93 signatures backing Lambert's performance.