In this study, specific binding affinity between vancomycin (VAN) conjugated 40-50 nm gold nanoparticles (Au NPs) implemented on a sensor chip and pentapeptide mimicking pathogenic VAN resistant Enterococcus (VRE) wall membrane was employed as nanosensor format based on localized surface plasmon resonance (LSPR). Two pentapeptides, which were also anchored to 5 nm Au NPs, having two different terminal sequences of d-Ala-d-Ala and d-Ala-d-Lac respectively were compared in specific binding affinity toward the VAN conjugated nanosensor. Binding affinity difference identified by the LSPR characteristic in the nanosensor was shown in absorbance intensity and absorption wavelength shift between the two different terminal pentapeptides. The absorbance intensity clearly indicated that the pentapeptide having terminal d-Ala-d-Lac showed less affinity toward VAN conjugated nanosensor chip than that having terminal d-Ala-d-Ala. It indicates that the VRE itself has relatively weak binding strength towards the VAN, which may be used as a probe molecule for the VRE. Therefore, in order to design a VRE sensor using the VAN affinity, an indirect LSPR method can be devised instead of direct LSPR method.