In this study, Bacillus spp. isolated from one unit of poultry waste in city of Miyaneh, Iran. The bacteria were cultured in a sterile culture containing feather. Among the five isolated Bacillus spp. one strain was found to produce keratinase using feathers as a sole carbon and nitrogen source that identified as B. subtilis based on morphological and biochemical characteristics and 16S rRNA sequence analysis. SDS-PAGE analysis illustrated a monomeric protein form with a molecular weight of 35 kDa similar to B. licheniformis described by Ana et al.