This study established an indirect ELISA-based serological diagnostic method for Lyme disease in mice, using whole-cell antigens of Borrelia garinii strain SZ. Through systematic optimization of experimental conditions, the optimal reaction system was determined as follows: antigen coating concentration of 0.2 μg/μl, serum dilution ratio of 1:200, and enzyme-labeled secondary antibody concentration of 1:2000. This method ensured high sensitivity (OD value > 0.8) while significantly reducing antigen consumption. Antibody dynamic analysis revealed that serum antibody levels in infected mice peaked between 12–25 days post-inoculation, with a specificity antibody ratio (ArB%) threshold of 41.7% to distinguish positive and negative samples. The study also evaluated cross-reactivity between B. garinii and Borrelia burgdorferi (B31) strain as well as Borrelia afzelii (BO23)strain, indicating certain cross-reactivity with BO23 but no significant cross-reactivity with B31. The results demonstrated that this method is simple, cost-effective, and suitable for primary laboratories and small-scale screening. It provides a reliable standardized technical support for the laboratory diagnosis and epidemiological surveillance of Lyme disease.