Cutin is a polymer that is constructed in plants by the condensation and oxidation of fatty acids and plays a key role in the protection of plants against pathogens. Cutinase is a hydrolase enzyme that breaks down the cutin. The purpose of this study was to extract cutin from red apples with oxalate buffer, cutinase enzyme activity assay in LB culture, and bioinformatic analysis. To attain these purposes the cutinase-producing strains that had previously been isolated were inoculated in culture medium containing cutin. After initial culture, the bacteria were cultured in LB medium and cutinase activity was measured using the p-Nitrophenyl butyrate. In order to execute bioinformatic analysis, the isolated sequences of six cutinase-producing bacteria were analyzed based on computational data bases and their phylogenetic trees were prepared. Then, the similarities in the sequences of a large number of cutinase-producing samples were analyzed by drawing the phylogenetic tree. The results showed the separation of cutinase-producing prokaryotes from cutinase-producing eukaryotes. Then, the sequence of 16S rDNA of these cutinase-producing samples as well as the samples we had prepared were evaluated and their phylogenetic relationships were determined. This analysis showed that the new sequence stood alongside the bacterial samples. Thus, our cutinases may be similar with these bacterial cutinases in structure and function.