In order to achieve rapid and in situ detection of active bacteria, inorganic alkaline hydrogen peroxide was used as a “clean” reducing agent to reduce and prepare a kind of rambutan-like Au@Ag alloy nanoparticles, which was used as surface enhanced Raman scattering (SERS) substrate material. The results showed that the constructed rambutan-like Au@Ag alloy nanoparticles had high surface roughness and dense surface nano burrs, and the structure had strong adsorption ability on the surface of bacteria, and did not affect the biological activity of bacteria. The rambutan-like Au@Ag alloy nanoparticles have large number of “nano-tip” and “nano-gap” on the surface of single particles, which can produce strong local surface plasmon resonance effect. The rambutan-like Au@Ag alloy nanoparticles are adsorbed on the bacterial surface, which could effectively increase the SERS “hot spots” on the bacterial surface, and it was easy to operate in the actual detection. Using the above characteristics, under the excitation of 633 nm light, the SERS fingerprint spectrum signals of active bacteria could be effectively obtained. With high sensitivity and efficiency of detection, it could be widely used in the SERS sensing analysis of microbial cells.