To perform the mechanism study of special association for vancomycin and D-Ala-D-Ala-containing peptides on the interface of solution and self-assemble monolayer,the binding between vancomycin and pentapeptide(Lys-Lys-Gly-D-Ala-D-Ala) was investigated by flow injection surface plasmon resonance(FI-SPR) and flow injection quartz crystal microbalance(FI-QCM).To facilitate the formation of a compact vancomycin adsorbates layer with a uniform surface orientation,vancomycin molecules were attached onto a preformed alkanethiol self-assembled monolayer.By optimizing the conditions for the binding between Lys-Lys-Gly-D-Ala-D-Ala and vancomycin on the assembled chip,the detecting limit of Lys-Lys-Gly-D-Ala-D-Ala was greatly improved(reaching 0.5×10-6 mol/L or 7.5×10-12 mol).The equilibrium constant of the association of Lys-Lys-Gly-D-Ala-D-Ala with vancomycin was also obtained(KAds=5.0×104 L/mol).