Using a Dual-View wavelength splitter, a custom-made filter block and only a single intensified charge-coupled device (ICCD), a three-channel real-time fluorescence imaging method for single living cell research was established based on an ICCD fast fluorescence micro-imaging system. The relevant calibrating method for images was also developed to eliminate the influence introduced by spectral crosstalk. Double-labeled with two fluorescent indicators, Annexin V-FITC and SNARF-1, mouse thymocyte was treated with S-nitrosoglutathione (GSNO) and the relationship between their apoptosis and intracellular pH changes was studied in real-time at the single living cell level. The results not only showed the different regularities of intracellular pH changes between apoptosis induced by GSNO and spontaneous apoptosis, but also provided evidence for the application of the three-channel real-time fluorescence imaging method to bio-medical studies.