Bacterial Cholesterol oxidase (CHO) is a monomeric, bi-functional and Flavin adenine dinucleotide-linked enzyme that catalyzes oxidation and isomerization cholesterol in the presence of molecular oxygen. Recently, powerful pesticide properties of these enzymes have been discovered on pest and in agriculture was used as a new generation of biological pesticides in cotton plants. This study aimed to isolate and molecular identification of Streptomyces bacteria producing CHO from Lorestan soil to provide biological pesticide in future. A total of 100 samples were collected from garden and agricultural land soils of Lorestan province. First, samples were cultured in cholesterolcontaining medium as an alone carbon source. Identification of grown bacteria was performed using the morphological characteristics, biochemical and microbial tests. Then, the colorimetric and browning of culture medium confirmed the activity of CHO. Then, the activity of cholesterol oxides was assayed under various temperature and pH. Obtained strains were confirmed by amplification of 16s rRNA gene using PCR technique and sequencing. A total of three producing cholesterol oxidase bacterial samples were isolated. The results showed that obtained strains belong to Streptomyces. The activity of cholesterol oxidase enzyme was confirmed by the colorimetric, and browning of culture medium tests. Finally, the molecular identity of the bacterial species was detected by sequencing PCR products. The results showed that the bacterial species, Streptomyces, isolated from the soil is capable of producing cholesterol oxidase enzyme.