We have demonstrated successful two-photon excitation fluorescence bioimaging using a high-power pulsed all-semiconductor laser. Toward this purpose, we developed a pulsed light source consisting of a mode-locked laser diode and a two-stage diode laser amplifier. This pulsed light source provided optical pulses of 5 ps duration and having a maximum peak power of over 100 W at a wavelength of 800 nm and a repetition frequency of 500 MHz.