In order to establish a fast and accurate method for novel DMIs fungicide screening, lanosterol 14α-demethylase of Magnaporthe grisea expressed in E.coli was used as target enzyme and the DMI fungicides diniconazole, tebuconazole, triadimenol and triadimefon were used as representative fungicides，the effects of enzyme activity, enzyme purity and concentration on the binding spectra were investigated. The results showed that active enzyme, elimination of interference of other P450s and proper enzyme concentration were necessary for obtaining accurate binding spectra. The K_d values of diniconazole, tebuconazole, triadimenol and triadimefon were 0.143μmol/L, 0.24μmol/L, 0.257μmol/L and 0.307μmol/L respectively, which significantly correlated to their 120h-EC₅₀ values on the growth of Magnaporthe grisea. The results indicated that the binding spectra of fungicide and lanosterol 14α-demethylase can serve as a reliable and fast method for novel fungicide screening.