The use of a novel ammonium ion-specific copper-polyaniline nano-composite as transducer for hydrolase-based biosensors is proposed. In this work, a combination of creatinine deaminase and urease has been chosen as a model system to demonstrate the construction of urea and creatinine biosensors to illustrate the principle. Immobilisation of enzymes was shown to be a crucial step in the development of the biosensors; the use of glycerol and lactitol as stabilisers resulted in a significant improvement, especially in the case of the creatinine, of the operational stability of the biosensors (from few hours to at least 3 days). The developed biosensors exhibited high selectivity towards creatinine and urea. The sensitivity was found to be 85±3.4 mA M−1 cm−2 for the creatinine biosensor and 112±3.36 mA M−1 cm−2 for the urea biosensor, with apparent Michaelis–Menten constants (KM,app), obtained from the creatinine and urea calibration curves, of 0.163 mM for creatinine deaminase and 0.139 mM for urease, respectively. The biosensors responded linearly over the concentration range 1–125 µM, with a limit of detection of 0.5 µM and a response time of 15 s. The performance of the biosensors in a real sample matrix, serum, was evaluated and a good correlation with standard spectrophotometric clinical laboratory techniques was found.