The 'Everlasting Love' singer will portray Santana's (Naya Rivera) mother Maribel Lopez in the season three finale on May 22.
Speaking about her Glee cameo in a behind-the-scenes video, Estefan declared: "I am beyond thrilled because my daughter and I have been watching religiously from season one and I'm just so happy to be here.
"Naya [plays] my favourite character. I love her, I think she's phenomenal, she gets real good zingers in. I love to listen to the insults that she throws at everybody.
"She's got amazing chops, plus she's a great dancer. I love them all, I think they all bring something unique and special."
Asked what it was like working with Estefan, Rivera replied: "She's awesome, she's so sweet. She's super tiny and just like a ball of energy and really excited to be here."
The season finale of Glee, titled 'Goodbye', will be the last episode before the show splits into two settings - McKinley High and New York.
Jenna Ushkowitz, who plays Tina, described the episode as "the end of an era", while various cast members including Amber Riley (Mercedes) and Dianna Agron (Quinn) are rumoured to be leaving the show.
Glee will conclude on Tuesday (May 22) at 9/8c on Fox. In the UK, the show continues Thursdays at 8pm on Sky1.
Watch a sneak peek of Gloria Estefan's Glee appearance below: