The cyp51 primers and two pairs of mutant primers which removed different transmembrane region were designed based on Ustilago maydis cyp51 gene structure analysis. The full cyp51 DNA fragment as well as mutant cyp51 genes were amplified and cloned by using the total DNA from Ustilago maydis as template, then subcloned into different expression vectors. The recombinant expression plasmids were transformed into Escherichia coli BL21 (DE3), BL21 (DE3) pLysS and Rosetta (DE3) respectively. A series of experiments leads to the finding that only pET32-YH-35 could be highly expressed at the optimal condition of 30oC induced with 0.5 mmol/L IPTG. The expressed protein (CYP51) showed biological activity by spectra analysis of the protein binding to 4 standard fungicides and to 14 XF-synthetic fungicide compounds, and only one XF-synthetic fungicide compound (XF-113) was similar to standard fungicides in binding constant. This compound is promising to be a new effective antifungal drug. These results will facilitate the further study on the mechanism of pathogenic fungi CYP51 and pesticide molecules, and will provide a new idea for efficient design and development of new anti-fungal drugs.