Abstract: A biosensor for the determination of fructose was constructed by the immobilization of the enzyme fructose dehydrogenase (FDH) behind a thin non-conducting electropolymer film of 1,3 phenylene diamine-resorcinol. Two different types of electrochemical mediators, soluble hexacyanoferrate (III) (FeCN) and the water insoluble tetracyanoquinodimethane (TCNQ) were applied as redox mediators for the amperometric measurement of fructose. Optimization of the biosensor was done with respect to operating conditions by varying the operating parameters such as pH, temperature, mediator concentration, and enzyme loading. The optimized biosensor was found to have a minimum detection limit of 10 µM with a linear range upto 1.0 mM. The performance of the two mediators was also compared with respect to stability. A simple, lumped parameter mathematical model was proposed to describe the steady state response of the biosensor and experimentally verified with the constructed biosensors.