[Background] Serine protease plays an important role in the biological control of Trichoderma spp. [Objective] This study provided theoretical support for the development of the protease biocontrol preparation and genetic modification by studying the serine protease S8/S53 superfamily gene information and biological functions of Trichoderma virens. [Methods] Twenty-three serine protease genes were identified from T. virens Gv29-8 genome by bioinformatics analysis. Four serine protease genes identified from Arthrobotrys oligospora ATCC 24927 genome were used as controls. The characteristics, protein structure, evolutionary status and function of 27 serine protease genes were predicted and analyzed. [Results] The 27 genes were significantly different in structure, and the encoded proteins had typical serine protease catalytic triad structure, belonging to the S8/S53 superfamily, which were divided into 6 subfamilies. The conserved domain of proteases in the same subfamily was similar in length, with high similarity and conservative sequences near the catalytic residues. Phylogenetic analysis showed that the serine proteases of the same subfamily were clustered into one group. [Conclusion] Some serine protease genes of T. virens and A. oligospora have strong similarities in structure and protein properties and close genetic relationship, both belonging to the S8_PCSK9_ProteinaseK_like subfamily. It is speculated that T. virens and A. oligospora have similar functions of serine protease, which can inhibit plant pathogenic fungi and degrade the body wall of nematodes.