Lyme disease is one of the most common vector-borne infectious diseases globally, partly due to the absence of a vaccine for humans. Hence, in this study, an immunoinformatics method was used to design a multi-epitope vaccine (MEV) against Borrelia burgdorferi. The optimal B- and T-cell epitopes from Borrelia burgdorferi proteins (BmpA and OspC) were joined with the appropriate linkers to construct a MEV. In addition, beta-defensin was included as an adjuvant in the vaccine construct. Secondary and tertiary structures of MEV were predicted, refined and validated. The developed vaccine was high antigenicity, non-allergenicity, solubility and stability. The Ramachandran plot, ProSA-web and ERRAT were employed to ensure the final model''s authenticity. The immune simulation confirmed acceptable responses of both cellular and humoral immune. The vaccine''s binding stability with Toll-like receptor 2 (TLR2) was confirmed using molecular docking and molecular dynamics (MD) simulation. Furthermore, MEV effectively stimulated high-level antibody production in mice, significantly promoted splenocyte proliferation in immunized mice, and markedly enhanced splenic IFN-gamma and IL-4 mRNA transcription levels. These results suggest that MEV, as a novel vaccine candidate, holds significant potential for future prevention and control of Borrelia burgdorferi infections.