Cholesterol oxidase which oxidizes cholesterol with oxygen to give cholestenone and hydrogen peroxide, is prepared by culturing Nocardia erthropolis ATCC 17895, Nocardia erthropolis ATCC 4277, Nocardia formica ATCC 14811 or Proactinomyce erythropolis NCIB 9158 first on a peptone-containing mineral salt medium and thereafter on at least one sterol compound as the sole source of carbon, and then isolating the cholesterol oxidase formed from the cultured micro-organism, wherein said sterol compound is of the formula ##STR1## in which R.sub.1 and R.sub.2 are hydrogen or together represent a double bond,R.sub.3 and R.sub.4 are hydrogen or together represent a double bond, andR.sub.5 is hydrogen or alkyl containing up to 3 carbon atoms.