Aims To provide information on the time‐dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis. Methods and Results For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4′,6‐diamidino‐2‐phenylindole) and Auramine‐O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1 s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1 s with increasing DAPI concentration, whereas it remained constant for 2–3 s with increasing Auramine‐O concentration, for example at a yeast concentration of 100 µg ml−1. Conclusions For the conditions of yeast >10 µg ml−1, DAPI >1 µg ml−1 and Auramine‐O >0·1 µg ml−1, τ could be adjusted to below 5 s to achieve a rapid and stable staining. Significance and Impact of the Study Design and operating conditions for rapid and online measurements of microbes can be optimized.