Pulsed and continuous-wave (CW) lasers have been widely used as the light sources for photodynamic therapy (PDT) treatment. Singlet oxygen (¹O₂) is known to be a major cytotoxic agent in type-II PDT and can be directly detected by its near-infrared luminescence at 1270 nm. As compared to CW laser excitation, the effects of pulse width and repetition rate of pulsed laser on the kinetics and production of ¹O₂ luminescence were quantitatively studied during photosensitization of Rose Bengal. Significant difference in kinetics of ¹O₂ luminescence was found under the excitation with various pulse widths of nanosecond, microsecond and CW irradiation with power of 20mW. The peak intensity and duration of ¹O₂ production varied with the pulse widths for pulsed laser excitation, while the ¹O₂ was generated continuously and its production reached a steady state with CW excitation. However, no significant difference (P > 0:05) in integral ¹O₂ production was observed. The results suggest that the PDT efficacy using pulsed laser may be identical to the CW laser with the same wavelength and the same average fluence rate below a threshold in solution.