Trichoderma hamatumis a filamentous fungus that serves as a biological control agent for multiple phytopathogens and as an important resource promising for fungicides. However, the lack of adequate knockout technologies has hindered gene function and biocontrol mechanism research of this species. This study obtained a genome assembly ofT. hamatumT21, with a 41.4 Mb genome sequence comprising 8170 genes. Based on genomic information, we established a CRISPR/Cas9 system with dual sgRNAs targets and dual screening markers. CRISPR/Cas9 plasmid and donor DNA recombinant plasmid were constructed for disruption of theThpyr4andThpks1genes. The result indicates the consistency between phenotypic characterization and molecular identification of the knockout strains. The knockout efficiencies ofThpyr4andThpks1were 100% and 89.1%, respectively. Moreover, sequencing revealed fragment deletions between dual sgRNA target sites or GFP gene insertions presented in knockout strains. The situations were caused by different DNA repair mechanisms, nonhomologous end joining (NHEJ), and homologous recombination (HR). Overall, we have successfully constructed an efficient and convenient CRISPR/Cas9 system inT. hamatumfor the first time, which has important scientific significance and application value for studies on functional genomics ofTrichodermaand other filamentous fungi.