A novel colorimetric biosensor was explored for fast, sensitive, and on-site detection of Salmonella on a microfluidic chip employing immune gold@platinum nanoparticles (Au@Pt NPs) for specific bacterial labeling, a finger-driven mixer with two serial air chambers for efficient immunoreaction and a nuclear track membrane as specific-size microfilter for effective bacterial isolation from excessive immune Au@Pt NPs. First, the bacterial sample and immune Au@Pt NPs were pipetted into the mixing chamber and mixed sufficiently through repeated press-release actions on the small air chamber which could precisely control the air volume, leading to the formation of bacteria-immune Au@Pt NP conjugates. Then, the small and large air chambers were pressed simultaneously to push all the solution in the mixing chamber to flow through the microfilter for trap of the formed larger-size conjugates on the membrane and removal of the unbound smaller-size immune Au@Pt NPs. After the H2O2-TMB substrate was pipetted into the microfilter and catalyzed by the conjugates, ImageJ was used to analyze the color change for bacterial determination. This simple biosensor enabled Salmonella detection as low as 168 CFU/mL in 25 min.