BoAHV1 is a significant cattle pathogen, resulting in substantial economic losses worldwide. B-cell epitopes within the viral proteins are not well understood. In this study, we screened a 12-mer phage display peptide library using a commercial BoAHV1 antibody. A total of 16 phage clones displaying individual 12-mer peptides were identified as reactive with this antibody using dot bot analysis. Through sequence alignment, 10 putative BoAHV1 B-cell epitopes, designated as PV109, PV108, PV116, PV59, PV50, PV130, PV113, PV49, PV62, and PV133, located within viral proteins gB, gC, gG, gM, UL36, UL37, and UL49, respectively, were recognized by both commercial BoAHV1 antibody andBoAHV1 IgG positive cattle serum through dot blot assay. Interestingly, immunization of mice with the synthesized peptide PV116 led to the production of antibodies suitable for Western blot analysis. Furthermore, an ELISA kit for the detection of BoAHV1 IgG antibodies in serum was developed, utilizing the identified epitope PV49, PV108, PV109, and PV116 as coating antigen. Collectively, we have identified 10 novel B-cell epitopes ofBoAHV1. Among them, PV116 is capable of inducing the production of antibodies suitable for Western blotting assay, which also shows potential for the development diagnostic tools.