Conductometric microbiosensors for the determination of trypsin were elaborated via the modification of microfabricated interdigitated gold electrodes by a cross-linked urease/BSA coating covered by a gelatin film. The resulting microelectrodes were exposed to different trypsin concentrations ranging from 100 pg/mL to 1mg/mL (1 mU/mL to 10,000 U/mL) for selective proteolytic degradation of the gelatin film. Then, the conductometric response of the microbiosensors to urea (33 microM) was recorded as a function of the trypsin concentration, the gelatin amount (8-80 ng) and the incubation time (40s, 100 min). The optimum incubation time for each trypsin concentration was determined leading to a detection limit of 100 pg/mL (1 mU/mL). In these optimized conditions, the proof of concept of this sensitive, disposable, low-cost and label-free trypsin biosensors based on a conductometric transducer was demonstrated for the first time.