The antibody response of serum IgM and IgG of patients with neuroborreliosis and erythema migrans of Lyme borreliosis (LB) was examined against a 41-kDa flagellar antigen and an 8-mer synthetic OspC8 peptide (VAESPKKP) derived from the C-terminus of outer surface protein C (OspC) from Borrelia garinii. We developed a streptavidin-modified biochip-based immunodiagnosis and compared it with conventional methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). The diagnostic sensitivity of the coated biochips was demonstrated to be identical, and the results of conventional assays such as ELISA and WB were confirmed. Flagellar antigens lead to better diagnosis because of a higher discriminative value. By contrast, OspC8, a peptide derived from the outer surface antigen, is less sensitive to identify immunity in LB. The inferior antigenicity of OspC8 may be due to epitope masking. Overall, this system is open to simultaneously analyze a larger family of peptides differing in length. Thus, an array approach is generally more advantageous to extend the pattern of antigens to be tested for antigenicity in LB. Serial analysis during ongoing disease may be valuable to learn more about the course of the disease and intermittent reactivation of infection. Protein biochip as a potential substitution of ELISA and WB method offers the opportunity to study serum immunity in a multiplicity of patients simultaneously.