Putrescine uses for the daily functions of living cells as a biogenic amine in small amounts and is available in protein-rich foods such as fish, cheese, and so on. The overconsumption of these nutrients can lead to some complications in the human body. Therefore, the recognition of putrescine by effective methods at low concentrations is a matter to be considered in protein-rich foods. This research article describes a novel biosensor for the detection of putrescine based on N,N-dimethylenediamine units bearing pillar [5] arene derivative (P5-DMEDA) and diamine oxidase (DAOx). The working parameters such as the amount of P5-DMEDA or DAOx and pH were optimized to determine the optimum conditions. The analytical calibration curve for the putrescine biosensor was prepared with putrescine concentrations ranging from 0.016 to 0.143 μM (R2 =0.9936) with a detection limit of 016 μM. The developed biosensor was used to determine the putrescine levels in the fish sample and satisfactory recovery results (96.0%-97.0%) were obtained. 1558-1748.