Microbial interactions are closely related to human health, and secreted signal molecules from bacteria determine the gene expression of bacteria following bacterial cell density and signal molecule density. However, the conventional quantitative analysis of the number of bacteria requires several days using standard cultivation methods, and the detection of molecules secreted via microbial interactions is difficult since they are in extremely small amounts. In this study, we performed local fluorescence spectroscopy to quantitatively evaluate the density of the assembly of dispersoids (fluorescent microparticles and bacteria) under optical condensation at a solid–liquid interface on our developed bubble-mimetic substrate, which exhibits extremely low thermal damage after a few minutes of laser irradiation. The obtained results showed that the fluorescence intensity spectrum was positively correlated with the concentration of dispersoids even when only several tens of assembled microparticles were observed. Furthermore, a calibration curve was obtained by plotting the integrated fluorescence intensity by integrating the fluorescence intensity spectrum over the observed wavelength, and the concentration of living bacteria was quantitatively analyzed. The clarified mechanism of local fluorescence spectroscopy under optical condensation will pave the way for rapid and precise analysis of bacteria and their secreted biomolecules labeled with fluorescent dye.