Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDCs LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance.