Boris (almost) never outright talks about her gender in the story, it's i guess more "implied" than explicitly shown, maybe except when she has more personal conversations with Fedya later on. So, it's not like anyone who lives around her acknowledges it, she didn't even understand it herself for a looong long time. I won't deny she was made to present more femininely by Matthias himself since birth, mostly because of the weird association of Boris with the twins' dead mom Yelena. However, this didn't make Matthias acknowledge her identity. I doubt he would be nice about it even if Boris understood her own identity/feelings and was open about it with Matthias. To Matthias, Boris was his "son" who just looked like a girl to outsiders. People also generally referred to her as how they perceived her (you won't see Fedya calling Boris his sister for a long time, causing confusion to those who perceived her as a little girl). Basically it wasn't something anyone understood let alone discussed. In another universes where they could bring this up more easily, like my already existing human AU, Matthias seems supportive of Boris and her transition, but even then he's very disrespectful to her, mainly using approval and acknowledgement as a grooming tactic against Boris.
In short, he's not only homophobic to Fedya, Anıl (and himself) but also transphobic to Boris. It mostly stems from self hatred he's not even aware he has, though even if he %100 loved himself he would still be a piece of shit to everyone around him.