Chitosan oligosaccharides (COS), as an important biological functional component, are mainly produced from marine resources, while their source face great challenges, such as seasonal variation, discontinuity and demineralization. In this study, the chitosan was extracted by alkaline method from Trichoderma asperellum, and then it was degraded with enzymatic hydrolysis to obtain the COS. The physicochemical characterization of chitosan from Trichoderma asperellum was compared with commercial chitosan, including chitosan content, deacetylation degree, chemical group, and molecular weight. Then, the enzymatic hydrolysis rates of Trichoderma asperellum chitosan and commercial chitosan were similar, 89.37 and 91.56%, respectively. Moreover, the composition of COS was tested by TLC, HPLC and MALDI-TOF, which results shown that Trichoderma asperellum COS was composed of chitotriose, chitopentaose and chitohexaose, and commercial COS was composed of chitotriose, chitotetraose, chitopentaose, chitohexaose and four acetylated chitohexaose. The antibacterial activity for Trichoderma asperellum COS was greatly stronger commercial COS. In conclusion, Trichoderma asperellum proved to be a novel potential source for COS production.