At present, the scenario of textile industry has been radically changed to use eco-friendly chemicals and enzymes for various wet processing of wool fibres. In general, wool bio-polishing is not so easy because of its complex fibre structure. The wool fibre has about 18 amino acids with the major composition part that contains proteins (97%) and lipids (1%). The wool fibre has some intrinsic properties (due to its morphological and scale structure) that makes wool fibre tendency to felting and shrinkage. Many research works have been carried out on bio-polishing of wool fibres using various enzymes. In this research work, we made an attempt to synthesize keratinase enzymes for polishing of wool fibres. This is because keratinase enzyme has the ability to attack the disulfide (-S-S-) bond in the keratin substrate, so it can be employed for bio-polishing of wool. The keratinase enzymes were produced by taking sheep wool and soil from sheep cattle through various biological processes. The enzymes produced through the mass culture and the enzymatic activity were observed through keratinase activity assay testing. It is reported that the enzyme production is realized better to the source wool fibre and cattle sand taken. The optimized production process may be the better alternative way for bulk production of keratinase enzyme for bio-polishing of wool fibres. © 2019 World Research Association. All rights reserved.