Cholesterol oxidase (ChOx) has been immobilized onto zinc oxide (ZnO) nanoporous thin films grown on gold surface. A preferred $c$ -axis oriented ZnO thin film with porous surface morphology has been fabricated by rf sputtering under high pressure. Optical studies and cyclic voltammetric measurements show that the $\mathrm{ChOx}∕\mathrm{Zn}\mathrm{O}∕\mathrm{Au}$ bioelectrode is sensitive to the detection of cholesterol in $25–400\mathrm{mg}∕\mathrm{dl}$ range. A relatively low value of enzyme’s kinetic parameter (Michaelis-Menten constant) $\sim 2.1\mathrm{mM}$ indicates enhanced enzyme affinity of ChOx to cholesterol. The observed results show promising application of nanoporous ZnO thin film for biosensing application without any functionalization.