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This study explores the possibility of simultaneous and specific detection ofSalmonella serovars by surface plasmon resonance (SPR). The Plasmonic® SPR device wasused to develop this rapid assay. The sandwich immunoassay involves the use of apolyclonal anti-Salmonella antibody to simultaneous capture multiple Salmonella serovarspresent in a sample. This is followed by specific detection of the captured serovars usingO-specific anti-Salmonella antibodies. Milk spiked with Salmonella Typhimurium andSalmonella Enteritidis was used as a model system to establish the assay. The assay wasfurther extended to sequentially differentiate between the two Salmonella serovars on asingle SPR chip in a single channel. The assay was proved to work without any additionaldilution or clean-up steps. The sample volume requirement for the assay is only 10 μL. Thelower limits of detection for Salmonella Typhimurium and Salmonella Enteritidis were2.50×105 cells mL-1 and 2.50×108 cells mL-1, respectively.