DeSantis, 41, was in good spirits as she took the stage at the Hernando County Republican Party Lincoln-Reagan Day Dinner.
'I hear when you get a standing ovation before you even walk in the door you should probably just leave,' she joked after accepting the microphone from her husband, Gov. Ron DeSantis.
The governor revealed that his wife had been diagnosed with breast cancer in an emotional statement released October 4.
'Probably the most important person that has stood by me is our wonderful first lady,' the Republican told the crowd Saturday before revealing that she was in attendance.
'I trust in the Lord, I know how tough she is. She's doing very well. In fact, she's doing so well that she wanted to come here tonight and say hello to you, so here she is.'
The crowd greeted the First Lady with a standing ovation.
DeSantis, who shares three children with the governor, delivered a message of perseverance in a one minute, 30-second video obtained by Fox News.
'I think there’s a lot that we can be upset about, that we can be sad about: The direction of our country, the future of our nation, our families, our communities, our safety, our children, their education, their futures, our health and our wellbeing,' the First Lady proposed.
'But I'll tell you one thing for sure. I’m sure as hell not giving up,' she said, attracting more cheers from the rapt audience before looking over her shoulder at her husband.
'Never ever ever give up the fight, ever.'
Hernando County, the site of Saturday's dinner, is part of the Tampa-St. Petersburg-Clearwater metropolitan statistical area.
Gov. DeSantis, 43, announced his wife's diagnosis earlier this month.
'I am saddened to report that Florida's esteemed First Lady and my beloved wife have been diagnosed with breast cancer. As the mother of three young children, Casey is the centerpiece of our family and has made an impact on the lives of countless Floridians through her initiatives as First Lady,' the governor said in a statement.
'As she faces the most difficult test of her life, she will not only have my unwavering support but the support of our entire family, as well as the prayers and well wishes from Floridians across our state. Casey is a true fighter, and she will never, never, never give up.'
Casey DeSantis is a native of Troy, Ohio. She earned a degree in economics from the College of Charleston, according to her State of Florida biography. She's a three-time National Champion equestrian, as well as a runner-up Division I NCAA Champion.