Abstract: Feathers are major byproduct waste of poultry industries and produced in large amount. Keratin can be biodegraded by keratinolytic microorganisms and in this study, 45 keratinase producing actinomycetes and their enzyme production was isolated from hot springs, Rajgir, India. After Screening of keratinase enzyme production, the strain showed highest activity was selected and identified as Streptomyces sclerotialus by using polyphasic taxonomy including morphological and biochemical characteristics followed by 16S rDNA and DNA directed RNA polymerase beta subunit (rpo β) gene sequencing (accession no: HQ827802 and HQ827815). The effect of various substrates, temperature, pH and protease inhibitors, reducing agents and metals ions supplements in the production medium on enzyme production was studied and found that starch (3.5%) at 120 h, gives maximum activity followed by, protease peptone (1.5%) at 144 h recorded. The optimum temperature and pH recorded 55°C and 9.0, respectively. The enzyme was stable at 55°C for 1 h. Metals like MgSO4, CaCl2 were found strong inhibitory effect; whereas, Na2SO3 found to be stimulatory for enzyme production. Gel electrophoresis analysis revealed that the crude enzyme was monomeric with only a single protein band of 46 KDa were observed after ammonium persulfate precipitation.