The antimicrobial resistance of food-borne pathogenic bacteria, including Vibrio parahaemolyticus, has been reported globally, warranting the need to identify promising alternative antibiotics such as endolysins that originate from bacteriophages. In our previous study, we characterized a bacteriophage infecting V. parahaemolyticus, vB_VpaP_KF2, at the molecular level. In this study, an open reading frame encoding putative endolysin was cloned from the complete genome data and expressed in the Escherichia coli expression system. The recombinant endolysin, vB_VpaP_KF2_Lys, exhibited a novel lytic property against Gram-negative bacteria regardless of pretreatment with an outer-membrane permeabilizer. It was also stable over a wide range of temperatures, pH, and NaCl concentrations, and its hydrolytic spectrum was broader than that of the parent bacteriophage. From the results, vB_VpaP_KF2_Lys could be used as a biocontrol agent against food-borne pathogens in the field of food safety.