Recently, using bacteriophages as new molecular probes in reliable platforms for the detection of bacterial pathogens has attracted more and more increasing attentions. In this paper, a novel isolated Myoviridae bacteriophage SEP37 was covalently immobilized onto gold nanoparticles (AuNPs) modified gold disk electrode (GDE) surfaces using cysteamine (Cys) as a crosslinker. Substrates of GDE-AuNPs-Cys-Phage SEP37 and specific capture of Salmonella cells had been characterized using scanning electron microscopy (SEM) separately. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to study the electrochemical response of the biosensor interface manufacturing and bacterial capture process. Under the optimal experimental conditions, this phage-based EIS biosensor was able to detect Salmonella with a wide linear range from 2 × 10 to 2 × 10 colony forming unit (CFU)/mL within 30 min in spiked lake water and lettuce samples, with a limit of detection (LOD) of 17 CFU/mL. The detection linear range of spiked chicken samples was 2 × 10 to 2 × 10 CFU/mL, with a LOD of 1.3 × 10 CFU/mL. In combination with a pre-enrichment process for 3.5 h, this assay could reach a LOD of 1 CFU/mL in chicken breast meat samples. Besides, this phage-based EIS biosensor provided good reproducibility and stability. This phage-based EIS biosensor opens a new opportunity for the detection of pathogenic bacteria using the inherent selectivity of bacteriophage recognition.