[Objective] Identification the chemical structure of chitinase GcCHI1 that isolated and purified form Gliocladium catenulatum HL-1-1 strain, and tested the antifungal effects and revealed the antifungal mechanism on several kinds of plant pathogenic fungi. [Methods] Nano-ESI-MS/MS methods and Oxford cup method. [Results] Peptide mass fingerprint atlas of trypsin hydrolysis peptide, MS/MS atlas and 3 peptide amino acid sequences (<15 amino acids), called LYNSNDAIEAFISR, VIGYFTQWGIYGR and LNLGIGYYGR were received. After blasted in the Mascot database, GcCHI1 was found the highest homology similarity with chitinase A of Stenotrophomonas maltophilial strain 34S1. Another result about antifungal effects and antifungal mechanism found chitinase GcCHI1 inhibited hyphal elongation, spore germination and sclerotium germination of such strains as Rhizoctonia solani, Sclerotinia sclerotiorum, and Botrytis cinerea et al.. [Conclusion] This paper analyzed the chemical structure of chitinase GcCHI1. And revealed the antigungal mechanism of biocontrol fugus HL-1-1 for chitinase GcCHI1’s antifungal activities.