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Salmonella is a common pathogenic bacterium. Salmonella isolates have been sorted into different serogroups according to the specific lipopolysaccharide present in the somatic surface (O antigen) of the bacterium. The virulence and pathogenicity of Salmonella vary greatly between different serogroups. Salmonella of O:2, O:3, O:4, O:7, and O:9 serogroups contribute to a large share of Salmonella outbreaks. Traditional slide agglutination tests to classify Salmonella serogroups are tedious because of their low testing efficiency, complex operation, and subjective judging criteria. Immunochromatographic assays have been extensively described in the literature, which discussed their low cost, rapid detection, and portability in multitude of fields, such as agriculture and environmental sciences. In this study, a biosensing multiplexer based on immunochromatographic assay (bio-MUX) was designed for rapid and high-throughput classification of five Salmonella serogroups. The five tests for the serogroups (i.e., O:2, O:3, O:4, O:7, and O:9) were integrated in three test lines. The results can be exported rapidly in 15 min. The sensitivity and specificity for the five serogroups exceeded 96% and 97%, respectively. No expensive or large instruments were needed in the bio-MUX. With these advantages, the proposed bio-MUX deserves to be promoted and expanded for on-site circumstances and specialized laboratories.