The uricase-gold nanoparticles (AuNPs) composite nanomaterial was covalently immobilized on a biofilm, eggshell membrane. The uric acid biosensor was constructed by positioning the immobilized biofilm on the surface of a Clark dissolved oxygen electrode. The scanning electron micrograph revealed that the uricase-AuNPs composite nanomaterial was successfully immobilized. The effects of enzyme loading, pH, concentration of phosphate buffer and temperature on the biosensor response have been studied in detail. The results showed that response time of the biosensor was 60 s, the linear range for the detection of uric acid varied from 1.0 µM to ~1.0 mM with a detection limit of 0.8 µM (S/N = 3) and relative standard deviation of 2.6% for 0.1 mM uric acid (n = 6). Some common potential interferents in samples demonstrated no interferences. The biosensor has been successfully applied to determine the uric acid level in urine and serum samples.