Chitooligosaccharides (COS), an important biological functional component, are mainly extracted from marine products, but its raw materials are currently facing challenges such as marine resources pollution and demineralization. This study aimed to explore Trichoderma asperellum as a novel source to prepare COS. The COS were prepared by the enzymatic degradation of chitosan from T. asperellum, and single factor experiment and orthogonal designs were used to optimize the enzymatic conditions for the preparation of COS. The composition of COS was performed by thin‐layer chromatography, high‐performance liquid chromatography, and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. The results showed that the degree of deacetylation of T. asperellum chitosan was 87.59%, and its enzymatic hydrolysis yield was 89.37 % under optimized extraction conditions. Moreover, the composition of COS in T. asperellum included chitotriose, chitopentaose, and chitohexaose. Compared with shrimp shells, COS prepared from T. asperellum showed stronger antibacterial properties against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Salmonella bacilli. [ABSTRACT FROM AUTHOR]