We have investigated the characteristics of surface plasmon resonance (SPR) spectra as a function of metal thickness for the application of protein arrays by using a wavelength interrogation-based SPR biosensor (self-developed). Resonance intensity and wavelength were significantly affected by the thickness of Au film. Minimal resonance intensity was observed at a thickness of approximately 450 Å of the Au film and changed slightly according to the incidence angle. A Ti film as an adhesion layer had a significant influence on the half-width of the SPR spectrum and the resonance wavelength, and a 50Å-thick Ti layer under the 450Å-thick Au film yielded a good spectrum in these experiments. In addition, the interaction of anti-tissue transglutaminase and tissue transglutaminase was successfully analyzed on the array of Ti/Au (50 Å /450 Å) metal spots by using the wavelength interrogation-based SPR biosensor. Thus, it is important to consider the optimal thickness of the Au film and Ti adhesion layer when biospecific interactions are analyzed on protein arrays by SPR biosensors.