Abstract: In this study, the antifungal protein EuAFP1.2 gene of Eucommia ulmoides was cloned, its CDS sequence length was 924 bp, encoding a polypeptide with 307 amino acids. The plant overexpression vector pCAMBIA1301-35 S-EuAFP1.2 was constructed. The transgenic tobacco was genetically transformed, and 30 transgenic plants with EuAFP1.2 gene were screened. The fungal disease resistance of transgenic plants was compared with wild-type and empty-transformed tobacco plants. The results showed that the relative spot area of transgenic EuAFP1.2 plants was significantly smaller than that of wild-type and empty vector transformed plants after inoculation with Rhizopus microsporus for 6 d. SOD, POD and CAT activities of transgenic tobacco increased first and then decreased before and after inoculation, and were significantly higher than those of wild-type and transformed empty vector plants at 24 h after inoculation. However, MDA content decreased firstly and then increased, and decreased rapidly to 14.89 nmol/g at 24 h after inoculation, which was significantly lower than that of wild-type and transformed empty vector plants. The proline content of transgenic tobacco was significantly higher than that of wild-type and no-load transgenic plants before and after inoculation. Before inoculation, the expression levels of PR1 a and PR2 in transgenic EuAFP1.2 tobacco were 2.43 times and 1.2 times of that in wild-type and 2.63 times and 1.27 times of that in no-load tobacco, respectively, but there was no significant difference in the expression levels of PR5. The expression of PR1 a and PR2 genes in EuAFP1.2 was up-regulated at 24 h after inoculation, and was significantly higher than that in wild-type and no-load plants. PR5 gene was induced to express. These results indicated that EuAFP1.2 overexpression could reduce the area of tobacco plant disease spots, improve the ability of scavenging reactive oxygen species and the expression of disease course related protein genes in tobacco plants, thus enhancing the inhibition of plant to R. microsporus and improving the ability of plant to resist fungal diseases.