"I want to say Sean, I love you, from the moment that I laid eyes on you," the music icon said before performing "La Vie en Rose" at the star-studded event. "I just wish you'd stop smoking cigarettes."
While the couple were married from 1985 - 1989, they've been spending more time with each other in the last few months.
The Milk star has been making headlines after being spotted at several of the "Living For Love" singer's shows during her Rebel Heart World Tour. While rumors have speculated they have rekindled their long dormant relationship, this was the first time the former lovers have since shown any kind of PDA.
Madonna recently defended her ex in a defamation suit by claiming the actor never abused her during their tumultuous four-year marriage.
While its unclear if the former couple are more than just friends, both Madonna and Sean have had their fair share of controversy in the last few days.
Penn is undergoing questioning after interviewing fugitive Mexican drug cartel kingpin El Chapo for a Rolling Stone interview just prior to his capture last week.
Meanwhile the mother of four is currently fighting for custody of her eldest son Rocco with second ex-husband Guy Ritchie.
Although time will tell if Sean is making her Madjesty feel like a virgin again, it should be noted that she famously called him her "true love" in her groundbreaking 1991 concert documentary Truth or Dare. #ExpressYourself