ZnO thin film was deposited on gold coated glass prism by RF sputtering technique in glancing angle deposition (GLAD) configuration. The structural, morphological and optical properties of the deposited film were investigated using X-ray diffraction (XRD), Atomic Force Microscopy (AFM) and Fourier Transform Infrared (FTIR) Spectroscopy. ZnO coated Au prisms (ZnO/Au/prism) were used to excite surface plasmons in Kretschmann configuration at the Au- ZnO interface on a laboratory assembled Surface Plasmon Resonance (SPR) measurement setup. Cholesterol oxidase (ChOx) enzyme was immobilized on the ZnO/Au/prism structure by physical adsorption technique. Polydimethylsiloxane (PDMS) microchannels were fabricated over ChOx/ZnO/Au/prism system and various concentrations of cholesterol were passed over the sensor surface. The concentration of cholesterol was varied from 0.12 to 10.23 mM and the SPR reflectance curves were recorded in both static as well as dynamic modes demonstrating a high sensitivity of 0.36° mM<sup>-1</sup>.