Background Cholesterol oxidase biosensors have been applied to detect cholesterol level in different in serum and foods samples. Due to a wide range of industrial and clinical applications of microbial cholesterol oxidase, isolation and detection of new microbial source (s) of cholesterol oxidase are very important.Results Among the potential strains, Streptomyces sp. strain NEAE-94 was chosen and identified based on cultural, morphological and physiological characteristics; in addition to the 16S rRNA sequence which had been deposited in the GenBank database under the accession number KC354803. Cholesterol oxidase production in shake flask by Streptomyces anulatus strain NEAE-94 was optimized using response surface methodology. The production parameters were first screened using a Plackett-Burman design and the parameters with significant effects on the production of cholesterol oxidase were identified. Out of the fifteen factors screened, agitation speed, cholesterol and yeast extract concentration were selected due to significant positive effects on the production of cholesterol oxidase. The optimal levels of these variables and the effects of their mutual interactions on cholesterol oxidase production were determined using Box-Behnken design. The maximum cholesterol oxidase activity (27.31 U/mL) was achieved at cholesterol concentration (4 g/L), the agitation speed (150 rpm/min) and yeast extract concentration (5 g/L). In comparison with cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 before the application of Plackett-Burman design, the statistical optimization resulted in an increased production by 4.66 times.Conclusions The maximal cholesterol oxidase activity is obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K 2 HPO 4 1, FeSO 4 .7H 2 O 0.01, MgSO 4 .7H 2 O 0.5), pH 7, inoculum size 4% (v/v), temperature 37°C, agitation speed 150 rpm/min, medium volume 100 mL and incubation time 5 days.