A DNA electrochemical interface biosensor based on screen-printed carbon electrodes (SPCEs) holds promise for point-of-care testing (POCT) detection of pathogens in food safety. Nevertheless, SPCE commonly has a rough surface and suffers from a relatively low electron transfer rate, disorder of DNA capture probes (CPs), and the steric hindrance effect of target nucleic acid binding. These issues lead to a low sensitivity. Herein, a simple and rapid electrochemical biosensor based on N-doped porous carbon (NPC)-modified SPCE and freezing-directed DNA combined with catalyzed hairpin assembly (CHA) was constructed for the one-pot detection of pathogens in food samples without time-consuming growth cultures. The biosensor was constructed by SPCE modified with NPC for enhanced electrochemical properties, and the DNA CP designed for CHA was stably fixed on the electrode for a high hybridization efficiency. Moreover, the signals amplified by CHA enable the selective and sensitive detection of pathogens without washing steps. This one-pot method is simple and sensitive with a wide detection linear range of 10