To determine why chitinase has different antifungal activity on different pathogenic fungi in vitro, we purified recombinant rice chitinase from Pichia pastoris and investigated its antifungal activity against four fungi - Rhizopus stolonifer (Ehrenb. et Fr.)Vuill, Botrytis squamosa Walker, Pythium aphanidermatum (eds.)Fitzp, and Aspergillus niger van Tiegh. Scanning electron microscopy (SEM)and Fourier transform infrared spectroscopy (FTIR)were used to analyze the surface microstructure and proportion of chitin in the cell wall of the four fungi, respectively. The results showed that the chitinase exhibited different antifungal activities against the four fungi, which was directly correlated to the surface microstructure and the proportion of chitin in the fungal cell wall. It will help understanding the antifungal mechanism of the recombinant chitinase and further determining its application scope on crop protection and post-harvest storage of fruits and vegetables.