I'm defending alone as a Fiora Peerless, someone moves in and ambushes with Kha'Zix. Would Fiora get her 1v1 passive?

Yes, Fiora gets her doubled Might. Her "one-on-one" condition is checked only at the moment she's designated as the defender — not when the trigger resolves. Since you were defending alone when the opponent moved in, the condition was met and the trigger goes on the chain. Playing Kha'Zix via Ambush as a Reaction happens after the trigger is already on the chain, so it can't stop it. (FAQ #11178, #1231, #1102)