We report the development of a new technique for label-free microarray readout based on surface-enhanced Raman scattering (SERS). In doing so, an investigation on optimized SERS substrates for the application to in situ microorganism analysis by Raman microscopy was carried out. Chemically synthesized nanoparticles were successfully applied to an immunoassay for label-free detection of single microorganisms. In this way, species specific, reproducible, and strong SERS spectra were collected from different bacteria immobilized on a chip. Furthermore, quantitative analysis of the microorganisms was performed using Raman mapping. Unlike conventional SERS detection of bacteria, which requires dehydration prior to analysis, our system enables us to detect and quantify microorganisms in an aqueous environment in situ. Hence, the nondestructive analysis of living bacteria cells is possible. Moreover, the "whole-organism fingerprint" SERS spectra can be adopted for further chemical characterization of microorganisms.