Rucker was performing at the famed venue in Nashville, Tennesse when an announcer asked the singer whether he would accept a few questions from the audience.
Rucker agreed, and after talking to two female fans, Paisley made a surprise appearance by taking the microphone to ask his own question.
Paisley told his friend, "I have two questions... One, are you still the worst poker player in the world?... And two... Would you like to become the newest member of the Grand Ole Opry?"
Rucker was given a standing ovation from the crowd and wiped tears from his eyes as he told fans, "You guys are here for one of the most special nights of my life. Thank you for sharing it with me."
After the show, he told People.com, "I thought it was just Brad playing another joke. When he said those words, it was like a tape delay from his mouth to my ears and then my brain. It means so much to be accepted into this circle, and there's no one I would rather have asked me than Brad."
The honour means Rucker will become the first African-American musician to join the famed Opry since Charley Pride became a member in 1993.
He will be inducted on 16 October (12).