The 38-year-old appeared on stage at the O2 Arena in London alongside the cast such as Chris Colfer, who plays Kurt, and Amber Riley, who plays Mercedes.
Paltrow played the role of Holly Holliday on several episodes of the hit US TV show.
The actress sang a version of the Cee Lo Green track Forget You, which she had also performed on the show.
She appeared after Naya Rivera, who plays Santana Lopez, told the crowd she had been named the sexiest member of the Glee club.
Paltrow then came on to the stage and challenged her to a sing-off.