Lumpy skin disease (LSD) is an infectious cattle disease caused by the lumpy skin disease virus (LSDV), posing a serious threat to the livestock industry. This study aimed to identify specific immunogenic targets for differential diagnosis and potential vaccine development. Using a phage display library covering the entire LSDV proteome, we screened sera from naturally LSDV-infected cattle and those vaccinated with the live attenuated goatpox virus vaccine (GTPV AV41) to identify differential antibody-binding viral peptides, leading to the identification of peptides within the LSDV103 protein. A truncated recombinant LSDV103 protein (TrLSDV103) was expressed and showed strong reactivity with sera from LSDV-infected cattle, significantly higher than that with sera from GTPV-vaccinated cattle. An indirect enzyme-linked immunosorbent assay (iELISA) based on the TrLSDV103 protein was developed, with a cutoff value of 0.361, demonstrating the diagnostic specificity of 100% (95% CI: 90.11-100) and the diagnostic sensitivity of 86.67% (95% CI: 70.32-94.69). The lowest detection limit for positive serum was 1:6400, with no cross-reactivity with five other bovine pathogens. Among 210 serum samples tested, 11 were suspected of LSDV infection. Additionally, TrLSDV103 protein immunization in BALB/c mice induced strong humoral and cellular immune responses, including significantly elevated IgG levels and antibody titers up to 1:204800. Cytokine detection assays showed a significant increase in IFN-γ and IL-1β levels. Flow cytometry analysis further revealed a marked increase in CD3CD4 T cells. In conclusion, TrLSDV103 is a highly immunogenic protein with strong diagnostic specificity, showing great potential as a differential diagnostic antigen and vaccine candidate against LSDV.