The Bills were made aware of Alonso's injury on Tuesday, though the franchise did not disclose information about how or when it was sustained, despite a statement being released.
Alonso is now expected to be out of action for seven to nine months, effectively ruling him out of the upcoming campaign.
The brief statement from Bills general manager Doug Whaley read: "We have learned tonight that Kiko Alonso injured his knee while working out in Oregon.
"We do not have the details at this point, but early indications are that it may be a significant injury."
Alonso played every defensive snap of his debut year last season, ending with the third most tackles in the league while also finishing second in The AP's NFL defensive rookie of the year voting.
The 23-year-old had already undergone hip surgery this off-season to repair a torn labrum, but was expected to be fit to begin practice with the rest of the Bills roster on July 18.