A novel bacteriophage-targeted electrochemical biosensor designed for accurate and quantitative detection of live Salmonella in food samples is presented. The biosensor is simply constructed by electrostatic immobilizing bacteriophages on MXene-nanostructured electrodes. MXene, renowned for its high surface area, biocompatibility, and conductivity, serves as an ideal platform for bacteriophage immobilization. This allows for a high-density immobilization of bacteriophage particles, achieving approximately 71 pcs μm. Remarkably, the bacteriophages immobilized MXene nanostructured electrodes still maintain their viability and functionality, ensuring their effectiveness in pathogen detection. Therefore, the proposed biosensor exhibited enhanced sensitivity with a low limit of detection (LOD) of 5 CFU mL. Notably, the biosensor shows excellent specificity in the presence of other bacteria that commonly contaminate food and can distinguish live Salmonella from a mixed population. Furthermore, it is applicable in detecting live Salmonella in food samples, which highlights its potential in food safety monitoring. This biosensor offers simplicity, convenience, and suitability for resource-limited environments, making it a promising tool for on-site monitoring of foodborne pathogenic bacteria.