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Pristine and Se doped ZnO nanoparticles (NPs) were successfully synthesized by thermo-mechanical method and their structural, morphological and optical properties are characterized. Here in, a series of experiments were carried out where cholesterol is treated with same concentration of Pristine as well as Se doped ZnO NPs. Several Cholesterol oxidation products (COP’s) such as 25-hydroxycholesterol and 26-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol are formed as observed by HPLC analysis. The batch of 5 wt % Se doped ZnO NPs exhibited highest cholesterol degradation efficiency followed by pristine ZnO NPs and 2 wt % Se doped ZnO NPs. The peak area corresponding to 7-ketocholesterol and 25-hydroxycholesterol is found to be 200980 AU and 200986 AU respectively. The mechanism of cholesterol degradation was correlated with incorporation of oxygen vacancies due to Se doping, which were likely intermediate levels for transiting photoexcited charge carriers for generation of hydroxyl radicals. Further, hydroxyl radicals generated during interaction of ZnO nanoparticles with aqueous media have been determined using terephthallic acid assay and 2′, 7′-di-chlorofluorescein (DCF) assay.