Spotted catfish is highly perishable due to the liberation of ammonia due to the degradation of urea by bacterial enzyme urease, hence maintenance and monitoring of urease degradation is of utmost importance in fish freshness. We have developed a urease biosensor to assess the quality of spotted catfish by immobilizing urease on a pH electrode using sodiumalginate and calciumchloride solution. Enzymatic degradation of the urea into ammonia by immobilised urease results in change in the potentials across glass electrode that can be measure by a potentiometric transducer. Spotted catfish stored at 30 oC showed a linear relationship between the degree of urea, liberation of the ammonia, deterioration of the freshness and the development of potentials across the urease biosensor. Hence, urease biosensor is a reliable, simple, and rapidmethod for the measurement of freshness of the fish flesh with high urea.