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Urea analysis is of considerable interest in clinical, agricultural and environmental chemistry. Urea biosensor helps in on-site monitoring of urea. The aim of this study was to evaluate the potential use of nanocellulose reinforced composite film as substrate for urea biosensor. Nanocellulose was prepared from non-spinnable short staple cotton fibres by controlled microbial hydrolysis process. This was used as a substrate to immobilize urease enzyme and, subsequently used to reinforce the starch/polyvinyl alcohol biocomposite film. Nanocellulose acted as both carrier for the enzyme and reinforcing agent in the film. Use of 1.5% nanocellulose increased the tensile strength of the resultant film to the tune of 2.5-fold. About 68% immobilization efficiency of urease onto nanocellulose was observed in the reported process. The biosensor could detect the presence of urea linearly in the range of 10 to 1000 ppm concentration in water with a response time between 30 sec and 1 min. The storage lifetime was 2 months when stored in 4C with the activity more than 90%. The study revealed that the nanocomposite film could significantly improve the performance and storability of urea biosensor.