Federer, sidelined for almost two months after tearing his meniscus, was due to play Argentine Juan Martin del Potro who defeated the former world number one in the 2009 U.S. Open final.
"I feel bad for the tournament and the fans, as I have rarely ever had to withdraw at such short notice," the third-seeded Federer said in a statement.
"I have not felt great for a few days, and unfortunately it got worse in the last 24 hours. I was really excited to have my comeback in Miami but am in no condition to play. I tried to warm up this morning, but it was clear that I could not compete."
The 34-year-old Federer said on Thursday that he had injured his knee in a freak domestic accident after slipping while running a bath for his twin daughters following his semi-final defeat at the Australian Open at the start of 2016.
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Horacio Zeballos, also of Argentina, replaced 17-times grand slam champion Federer and posted a 6-4 6-4 win over del Porto.
Fifth-ranked Rafa Nadal said he was disappointed Federer had to withdraw.
"It's obvious that a tournament with Roger is always better," the Spaniard said. "It's good that it's a stomach problem and at the same time it is not the knee, so it's not a big deal. But it's obvious that it's terrible news for the tournament."
(Reporting by Larry Fine; Editing by Tony Jimenez and Steve Keating)
