The formation of a Prussian blue film under and above a polypyrrole film containing formate dehydrogenase and β-nicotinamide adenine dinucleotide has been investigated by electrochemical quartz crystal microbalance and cyclic voltammetry techniques. Two different deposition mechanisms of Prussian blue were identified and explained when the polypyrrole coated substrate electrode was used. Very stable current and frequency responses were obtained when the Prussian blue film was deposited under the polypyrrole film. This layered structure is potentially useful in solving the leakage problems of a biosensing system involves immobilised mediator. This advantage has been demonstrated using a formate biosensor fabricated with the enzyme, formate dehydrogenase, a co-factor, β-nicotinamide adenine dinucleotide and a Prussian blue mediator.