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In recent times, biodegradation of used hospital cotton by using the bacterial strain is a promising and interesting field attracting worldwide researchers. It was observed that biodegradation of biomedical waste like cotton etc., is becoming a huge problem for both developed as well as developing countries. To overcome this problem, bacterial species were isolated using serial dilution and agar plating methods from different soil sources. Pure colony or single colony was isolated by the streak plate method. The bacterial species were identified with the help of various physical characteristics, staining, and biochemical activities. Strain improvement was done by UV and EtBr to know the best culture for amylase production. Purification of the crude amylase was done by dialysis, and its activity was calculated by DNS assay. Protein concentration was measured by Lowery’s method. The purified enzyme was characterized for the effect of temperature, pH, activator, and inhibitors. The bacterial colonies were isolated and named as IS2015 01 to IS2015 16, the total enzyme activity of the purified enzyme was calculated to be 0.96 mg/ml, and the amount of protein in the purified sample was calculated to be 1.47 mg/ml. Amylase purified here was found stable in a pH range of 7 - 9 and temperature range of 28°C - 37°C. The activity was found to be enhanced under the influence of cations such as Ca2+, Mg2+ and retarded under the influence of anions such as EDTA and SDS. The isolated bacterial strain and enzyme were found effective in degrading the biomedical cotton waste. In conclusion, the isolated bacterial strain and enzyme may be effectively used to degrade the biomedical cotton.