The present report addresses the application of surface plasmon resonance (SPR) phenomenon for urea sensing. The former promises a high sensitivity, label-free detection, and real-time information by monitoring the refractive index change at the metal–dielectric interface. In the present report, a highly sensitive urea biosensor has been developed by integrating a ZnO thin film matrix with the SPR technique. Kretschmann configuration has been used to excite the surface plasmon (SP) modes at the ZnO–metal (gold) interface using an indigeneously developed table top SPR measurement setup. Urease (Urs), the urea-specific enzyme, has been immobilized on the surface of ZnO thin film by physical adsorption technique. The SPR reflectance curves were recorded for the prism/Au/ZnO/Urs system in angular interrogation mode with phosphate-buffered saline (PBS) solution as the liquid media. The SPR resonance angle is found to be shifted toward a lower angle from 49.1 to 42.0 deg with an increase in the urea concentration (0 to ) in the PBS solution. The developed sensor (prism/Au/ZnO/Urs) is found to be highly sensitive [ or ] with detection accuracy of .