The detection of foodborne pathogenic microorganisms is an essential issue in molecular diagnostics. Owing to their ability to detect and measure low analyte concentrations, fluorescence-based assays have been widely used in molecular diagnostics. However, conventional fluorescence-based assays require sophisticated optics systems, such as a specific light source and light filter. To overcome these limitations, in the present study, we developed an optical sensing system using a retroreflective Janus microparticle (RJP) as a signaling probe. Compared to fluorescent dyes, the advantage of RJPs is that they do not require complicated optic systems, as they can be observed using visible light without a filter. To confirm that RJPs can be used as a probe for molecular diagnostics, in the present study, Salmonella was detected using a biotinylated stem-loop DNA probe to capture the target gene DNA and a streptavidin-conjugated RJP (SA-RJP) as the detection molecule. When the target gene DNA was present at the sensing surface where the stem-loop DNA probe was immobilized, the biotinylated stem-loop DNA probe was stretched, exposing biotin, which can react with SA-RJP. Since the amount of exposed biotin increased according to the concentration of the applied target gene DNA, the number of RJPs observed on the sensing surface increased with the concentration of the target gene DNA. Consequently, the concentration of Salmonella could be quantitated by counting the number of observed RJPs. Using this system, Salmonella at concentrations ranging from 0 to 100 nM could be effectively analyzed with high sensitivity and selectivity, with the limit of detection of 2.48 pM.