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The objective of this study was to investigate the development of a surface plasmon resonance (SPR) sensor platform equipped with multiple channels for the simultaneous determination of methicillin-resistant <i>S. aureus</i> (MRSA), methicillin-susceptible <i>S. aureus</i> (MSSA) and vancomycin-resistant <i>Enterococcus</i> (VRE), and vancomycin-susceptible <i>Enterococcus</i> (VSE). Drug resistance of <i>S. aureus</i> strains against cefoxitin and <i>Enterococcus</i> strains against vancomycin were investigated both using the minimum inhibitory concentration method (MIC) assay and the SPR system equipped with single and multiple channels. The MIC values of MRSA and MSSA ranged from 32 &#181;g/mL to &gt;128 &#181;g/mL and from 1 &#181;g/mL to 4 &#181;g/mL, respectively. The MIC values of VRE and VSE were between 64 to &gt;128 &#181;g/mL and 2&#8722;4 &#181;g/mL, respectively. With the multiple-channel system, the angle shifts of MRSA, MSSA, VRE and VSE were found to be &#8722;0.030&#176; and &#8722;0.260&#176;, &#8722;0.010&#176; and &#8722;0.090&#176; respectively. The antibiotic-resistant and susceptible strains were distinguished within 3 h for <i>S. aureus</i> strains and within 6 h for <i>Enterococcus</i> strains.