To get a better optimization expression of the Ustilago maydis CYP51 (P450-14DM, UmCYP51) protein in E. coli BL21(DE3), the different lengths of UmCYP51 gene that lacked the coding region for the putative membrane-spanning segment of the N-terminus were truncated. The first one is the wild type, the second one with 20 amino acids (60 base pairs) in N-terminus was truncated and the third one with 35 amino acids (105 base pairs) was truncated. Then these genes were incorporated into different expression vectors (pET28, pET32 and pGEX-KG) to construct nine recombinant expression plasmids (pET28-Um, pET28-Um-20, pET28-Um-35,pET32-Um, pET32-Um-20, pET32-Um-35, pGEXKG-Um, pGEXKG-Um-20 and pGEXKG-Um-35). The expression of recombinant plasmids were performed using 0.5 mmol/L of isopropyl β-D-thiogalactoside (IPTG) at 30℃. The culture harvested every 2 h up to 8 h. It was found that only recombinant plasmid pET32-Um-35 was expressed in E. coli BL21(DE3). Codon usage database (http//:www.kazusa.or.jp/coden) was used for the analysis of rare codon and software RNAStructure 4.5 was employed to study the mRNA secondary structure of translation initiation region. The results showed that rare codons rate in UmCYP51 gene is only 4.63%, the Rosetta (DE3) strain expressing some rare codons is not suitable for the protein expression of UmCYP51. Only the lowest energy of mRNA structure for pET32-Um-35 can obtained protein expression. These results are compatible with the experiments. Moreover, to design novel antifungal compounds against UmCYP51, based on the recently determined X-ray crystal structure human CYP51, a three-dimensional structure model of UmCYP51 was built through homology modeling using MODELLER 9V7 program. After refinement of the energy minimization and MD simulation using GROMACS 4.0.3 package, the UmCYP51 model was evaluated by PROCHECK Ramachandran plot statistics that indicated the designed model was in good quality. Commercial fungicide tebuconazole was docked into the model protein using Autodock 4.2.3 program to form the binding pattern of inhibitor with UmCYP51. The docking conformation of tebuconazole in the active site of UmCYP51 showed that the N-4 of the triazole ring was bound to heme iron with a distance about 0.245 nm. The hydroxy group of tebuconazole formed hydrogen-bonding interaction with the oxygen atom of carbonyl group for Ala265 with a distance about 0.245 nm. The mechanism of inhibitory activity of tebuconazole against UmCYP51 obtained from this study could aid in designing new antifungal compounds targeting this enzyme.