The invention provides an anti-subgroup B avian leukosis virus (ALV-B) strain cell line and application thereof. The ALV-B env gene is inserted into an expression vector, an established transfer vector is expressed in a chick embryo fibroblast cell line DF-1, and Zeocin drug screening is performed to obtain the Zeocin-resistant chick embryo fibroblast cell line DF-1/B with stably expressed ALV-B env gene; and the collection number of the cell line is CCTCC No.C201609. The detection and antivirus infection analysis on the DF-1/B cell line detect that the ALV-B env protein expressed in the DF-1 cell line has superinfection resistance to the ALV-B virus and can resist the virus infection replication quantity with the titer of 104TCID50. The DF-1/B cell line can be used for differential diagnosis on infection of different subgroup avian leukosis viruses, and has obvious clinical diagnosis value and wide application prospects.