Langmuir–Blodgett (LB) films of polyaniline (PANI) grown onto ITO coated glass substrates were utilized for the fabrication of Uric acid biosensor for efficient detection of uric acid by immobilizing Uricase via EDC–NHS coupling. The modified electrodes were characterized by atomic force microscopy (AFM). The response characteristics after immobilization of uricase were studied using cyclic voltammetry and electrochemical impedance spectroscopy techniques. The uricase/PANI/ITO/glass bioelectrode studied by CV and EIS techniques revealed detection of uric acid in a wide range of 0.05 mM to 1.0 mM, covering the physiological range in blood. A low Michaelis–Menten constant (Km) of 0.21 mM indicates the higher affinity of immobilized Uricase towards its analyte (uric acid). The fabricated uric acid biosensor based on PANI LB films exhibits excellent sensitivity of 0.21 mA/mM with a response time of 4 s, good reproducibility, long shelf life (8 weeks) and high selectivity.