If Touko's line is an ad-lib, that would be the first time she's indicated that she knows Sayaka is in love with her, right?
No, the ad-lib would be aimed at Yuu. That line doesn't quite match anything the girlfriend says during the play, let alone what Touko knows of Sayaka.
Well, time will tell if the line means anything in particular at all outside of the the context of the play. As you say, Sayaka’s astonished expression suggests that it’s a surprise to her, but if so, the upshot of such an ad lib is hard to specify without more context.
I do suspect that Touko has at least some inkling of Sayaka’s feelings, though. (Again, an example of Nakatani’s “options open” narrative style: Touko might have no idea, or she might have known all along, and either one would be consistent with the previous story.)