Objective Monkey B virus(BV), also known as Cercopithecine herpesvirus 1,is an important zoonotic pathogen. According to the national standard, antibodies are detected using BV as an antigen. However, the preparation of BV antigen is very stricted due to biosafety issues. Therefore, in this study, we used alternative antigens to detect the BV antibody by serological assay and verified their specifity and sensitivity. Methods A total of 135 blood samples from rhesus monkeys were tested by two ELISA method (BV and HVP2) and enzyme immunosorbent assay (EIA)method. The positive and suspicious samples were verified by immuno-fluorescence assay (IFA), Western blot and immunoblotting technique using HSV-1 gC1 purified glycoprotein as an antigen. Results The positive rates of HVP2-ELISA, BV-ELISA and HSV-1-EIA were 32.6%, 37.8% and 34.8%, respectively. Consistant result of the three detection method accounted for 91.1% (123/135), and the positive result were confirmed by IFA And WB.There were 12 suspicious samples,in which 33.3% (4/12) were verified to be positive. Conclusions Compared with BV antigen, the sensitivity and specificity of the alternative antigen HSV-1 are moe close than HVP2. Positive and suspicious samples should be verified by several method to avoid missed detection.