Infection with the spirochete Borrelia burgdorferi leads to Lyme disease, the most common tick-borne disease in North America, Europe, and Asia. Currently, Lyme disease is diagnosed using a two-tiered approach of ELISA/immunofluorescence, followed by Western blot analysis. These assays measure serological immune response to the infection, namely levels of IgG or IgM antibodies that bind to B. burgdorferi antigens. However, the existing approach is non-quantitative, lacks sensitivity, and may contribute to delayed diagnosis. In this study, grating-coupled fluorescence plasmonics (GC-FP) was used for rapid, highly-multiplexed detection of antibodies that bind B. burgdorferi proteins in human and mouse blood serum. GC-FP is an optical plasmonic method that enables quantitative detection of molecular interactions and can be incorporated into microfluidic format for highly multiplexed testing. We have demonstrated that this technique allows us to use only three microliters of blood serum to quantitatively detect multiple target antibodies within 30 minutes. We have also shown that GC-FP is faster and more sensitive than the traditional two-tiered Lyme disease testing scheme, making it attractive for diagnostic purposes. This proof-of-concept study provides foundations to develop GC-FP as a highly sensitive diagnostic tool to enhance the efficiency of assessment for Lyme disease patients, which will ultimately improve treatment outcomes.