Cyclodextrin (CD) is a kind of cyclic oligosaccharides, which forms host-guest interactions with hydrophobic molecules and is widely applied in capillary electrophoresis and pharmaceutical engineering. In this study, we established a succinyl-β-CD modified gold biochip for improvement of seroimmunological detection sensitivity of Lyme disease. We found that the CD modified biochip platform presented a stronger affinity property for VlsE protein in conjugation with >0.000475 μg/mL of antigen immobilization concentration, which was sensitive enough for fluorescence based assay. Detection limit for anti-VlsE IgG antibody was 0.39 μg/mL. Specificity of VlsE assay on the succinyl-β-CD modified biochip was successfully confirmed by an immunological block assay. Furthermore, the correlation coefficient (R2) between the fluorescence values by the biochip and the OD values by ELISA assay was 0.904, indicating this biochip-based immunological assay might be a potential substitute with the ELISA-based approach. This biochip platform would be not suitable for loading of flagellin and OspC.