Bacillus cereus is widely distributed food-borne pathogenic bacterium. Due to the harmness to human hearth and the generation of multidrug-resistant B. cereus, it is urgent to develop novel antimicrobial agents. Phage and phage endolysin were taken as novel antimicrobial substance for their specific lytic activity against pathogenic bacteria. In this study, a Myoviridae family phage, designated as vB_BceM-HSE3, infecting the pathogenic multidrug-resistant B. cereus strain was isolated and characterized along with its endolysin. Phage vB_BceM-HSE3 can specially infect the B. cereus group strains, including B. cereus, B. anthracis, and B. thuringiensis, and exhibits high temperature and pH tolerance, which endow it with high potential for been used in controlling pathogenic B. cereus group strains. Genomic analysis reveals that vB_BceM-HSE3 is a novel phage and only shows extremely low genome similarity with available phage genome. Functional analysis of endolysin PlyHSE3 encoding by vB_BceM-HSE3 shows that PlyHSE3 exhibits broader lytic spectrum than the phage and can lyse all the tested B. cereus group strains as well as the tested pathogenic strain of P. aeruginosa. PlyHSE3 also shows broad temperature and pH tolerance, and can efficiently lyse B. cereus strain at temperature at 4 degrees C and higher than 45 degrees C, which indicating that PlyHSE3 might can be used in controlling food-borne B. cereus during both the cold storage of food and the stage after the heat treatment of food. The findings of this study enrich our understanding of phage diversity as well as providing resources for developing phage therapy.