A coxsackievirus B4 strain and application thereof are disclosed. The strain is named KM140-G01 and is preserved in China Center for Type Culture Collection (CCTCC) on Jun. 18, 2023, and the preservation number is CCTCC NO: V202356, the preservation address is Wuhan University, China. The strain is a humanized coxsackievirus B4 wild type single purified strain, and has strong virus replication capacity, good genetic stability and immunogenicity; the strain can be applied to the development of human coxsackievirus B4 vaccine production strains, and is suitable for the development of attenuated coxsackievirus B4 vaccine and inactivated coxsackievirus B4 vaccine; the method can also be used for establishing an infection model on Vero cells and suckling mice, and is used for CVB4 infection and pathogenic mechanism research, CVB4 vaccine evaluation and antiviral drug screening.