African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in the icosahedral capsid assembly. This protein has good antigenicity and is also a target for developing diagnostic tools for ASF. To understand the molecular basis for the antigenicity of pB602L, a procaryoticly expressed recombinant pB602L protein was produced and applied to immunize mice for producing monoclonal antibodies (mAbs). A total of eight mouse mAbs were obtained and their binding epitopes were screened by Western blot against overlapped polypeptides of pB602L. Three linear epitopes were identified and designated Epitope 1 (366ANRERYNY373), Epitope 2 (415GPDAPGLSI423), and Epitope 3 (498EMLNVPDD505). Based on their recognizing epitopes, the eight mAbs were placed to three groups: Group 1 (B2A1, B2F1 and B2D10), Group 2 (B2H10, B2B2, B2D8, and B2A3), and Group 3 (B2E12), accordingly. mAbs B2A1, B2H10 and B2E12 were applied to detect pB602L in ASFV infected porcine alveolar macrophages (PAM) and pig tissues by indirect florescence assay (IFA), immunohistochemical staining, and immunogold labeling for electron microscopy, respectively. The results showed that pB602L was well detected with all the three mAbs by immunohistochemical staining and immunoelectron microscopy; but only B2H10 was suitable for detecting the protein in ASFV infected PAM cells by IFA. Taken together, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which provide biological materials and molecular basis for the basic and applied researches on ASFV.