Five different isolates of alkalophilic bacteria were obtained from water and sludge sample of Lonar lake ,in Buldhana district of Maharashtra . These isolates were identified as Cholesterol oxidase, Protease inhibitor, Amylase inhibitor. Alkaline Protease, Chitinase by 16S rDNA sequencing.  In this paper, the characters of Cholesterol oxidase produced by five most potent isolate were studied for its insecticidal potential. , Among the five isolates, four isolates (i.e A, B, C and D) were found to produce Cholesterol oxidase. Out of that, Bacillus thuringiensis serovar finitimus showed the highest (0.835 U mg protein^-1) COX inhibitory activity. The activity of COX was almost constant at wide range of pH (8-11).The optimum pH for the COX obtained from all the isolate was found to be at pH 10.The protein showed stability upto 1hr when incubated at pH 9 to 10 and stability of enzymes decrease at extreme pH-12. COX obtained from all the alkalophile isolates were optimally active at 50^oC.The thermal stability revealed that enzyme was stable at 30⁰C to 50 ^oC for upto 90 min. However ,the enzyme activity declined drastically at temperature higher than 50^oC.