The influence of laser irradiation on enzyme activity depends on the wavelength and energy of the light. The influence of wavelength and energy on enzyme activity is studied systematically, and whether laser and LED light sources have the same effect on enzyme activity is examined. Phospholipase C is exposed at 0~810 J/cm2 of lasers at the wavelength of 532, 808, 1064, 1342 nm and two LED sources at the wavelength of 640 nm and 810 nm respectively, and enzyme responses are evaluated by measuring ceramide concentration using high performance thin-layer chromatography (HPTLC) after irradiation of 0.5, 1, 2, 3, 4, 6, 17, 24 h. The duration of effect is evaluated from the experimental data. The enzyme activity can be increased by using either laser or LED source whose wavelength is located within a certain range, and the effect depends on the energy and wavelength of the light. The increase in enzyme activity continues for about 4 h after irradiation. The result shows that the duration of enzyme activity change should be included as one of the main laser parameters while reporting the effect of laser irradiation on enzymes. Besides, laser sources and LED sources have the same effect on enzyme activity with the same wavelength and absorbed energy.