I'd punctuate it as:
"It's been, what, twenty-five years since I've seen you?".
I don't think the "what" is, in itself, a question here. As dicitonary.reference.com says, it's an interjection:
interjection
- (used in exclamatory expressions, often followed by a question):
What, no salt?
Also the "twenty five years" isn't a question on it's own either. What we have is a statement posed as question: "It's been twenty-five years since I've seen you?" with the interjection "what" inserted in the middle.
To mark the pauses in speech either side of what, I'd use commas.