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Vanadium pentoxide (V2O5) was used as support material to immobilize the urease enzyme. V2O5 was prepared from a sol–gel process, and the immobilization of urease was done onto the surface of the PET/ITO/V2O5 film. Different concentrations of urea were tested to study the sensitivity of the biosensor. Tests with pH variation were carried out, and it was noticed that the total charge in the cyclic voltammograms decreases due to the increase in pH that influences the catalytic activity of the enzyme. From cyclic voltammograms, with a different scan rate, for linear correlation testing, it was observed that the anodic currents varied as a function of the scan rate. Based on the variation in the urea concentration, the current increased with the increase in the urea concentration, indicating the sensitivity of the material. The use of V2O5/urease showed favorable electrochemical responses for application in biosensors as well as being sensitive to the variation in the urea concentration and easy to obtain and prepare.