Recently we reported that gold nanoparticles (GNPs) with fully matched duplexes on their surfaces are selectively deposited onto walls of poly(dimethylsiloxane) (PDMS) microchannels at high salt concentrations. In this study, the surface plasmon resonance (SPR) imaging technique was applied to monitor this phenomenon for improvement of detection sensitivity and elucidation of the phenomenon. The microchip was fabricated by bonding a surface-patterned PDMS plate and a gold thin film-deposited glass substrate. Probe oligonucleotide-modified GNPs were hybridized with target oligonucleotides to make fully matched or single-base-mismatched duplexes. The hybridized GNP solution was mixed with an NaCl solution in a Y-shaped microchannel. The deposition of the GNPs onto the gold sensor surface was detected by SPR imaging. Discrimination of the targets was possible with limit of detection of 32 nM (19 fmol) without temperature control in 5 min. Detailed analysis indicated that a seed layer of GNPs was initially adsorbed onto the sensor surface regardless of the target sequence. Therefore, in combination with a portable SPR device, the proposed method is promising for point-of-care testing of single-nucleotide polymorphsims.