Since Salmonella Enteritidis is one of the major foodborne pathogens, on-site applicable rapid detection methods have been required for its control. The purpose of this study was to isolate and purify S. Enteritidis-specific phage (KFS-SE2 phage) from an eel farm and to investigate its feasibility as a novel, efficient, and reliable bio-receptor for its employment. KFS-SE2 phage was successfully isolated at a high concentration of (2.31 ± 0.43) × 1011 PFU/ml, and consisted of an icosahedral head of 65.44 ± 10.08 nm with a non-contractile tail of 135.21 ± 12.41 nm. The morphological and phylogenetic analysis confirmed that it belongs to the Pis4avirus genus in the family of Siphoviridae. KFS-SE2 genome consisted of 48,608 bp with 45.7% of GC content. Genome analysis represented KFS-SE2 to have distinctive characteristics as a novel phage. Comparative analysis of KFS-SE2 phage with closely related strains confirmed its novelty by the presence of unique proteins. KFS-SE2 phage exhibited excellent specificity to S. Enteritidis and was stable under the temperature range of 4 to 50°C and pH of 3 to 11 (P < 0.05). The latent time was determined to be 20 min. Overall, a new lytic KFS-SE2 phage was successfully isolated from the environment at a high concentration and the excellent feasibility of KFS-SE2 phage was demonstrated as a new bio-receptor for S. Enteritidis detection.