Putrescine oxidase (PUO) from Micrococcus rubens has been characterized, as a biocatalytic component for the development of electrochemical biosensors for the determination of putrescine. PUO was immobilized by cross-linking on screen-printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as mediator (TTF/SPCEs). Isothermal titration calorimetry was used for investigation of kinetic properties of immobilized PUO. These PUO/TTF/SPCEs showed a linear response range from 10 a 74μM, a capability of detection of 10.1 ± 0.6μM (n = 4, α = β = 0.05) and a precision of 6.7%. Octopus and zucchini samples have been analysed using HPLC as a reference technique.