The invention discloses an enterococcus exopolysaccharide with an immunomodulatory effect as well as a preparation method and application of enterococcus exopolysaccharide; enterococcus hiraei WEHI01 is used for fermentation to generate the exopolysaccharide, the exopolysaccharide is separated and purified, and the enterococcus hiraei WEHI01 is preserved in the China Center for Type Culture Collection, and has the preservation number of CCTCC NO:M 2019068. The prepared enterococcus exopolysaccharide comprises four components: EPS-I01, EPS-I02, EPS-I03 and EPS-I04, the average molecular weight of the component EPS-I02 is 2.28*10<4> Da, and the component EPS-I02 is composed of rhamnose, trehalose, arabinose, xylose, mannose, glucose and galactose according to the molar ratio of 1:1.76:5.48:1.76:2.52:6.2:20.92. The enterococcus exopolysaccharide prepared by the preparation method has an immunoregulation effect, can significantly improve the proliferation rate and phagocytic ability of macrophages, and significantly promote macrophages to secrete NO and cell factors, and the enterococcus exopolysaccharide as an additive is applied to the fields of food and medicine, and has a wide application prospect.