The present invention provides a method and system for manufacturing a microstructure in a photosensitive glass substrate, which include the steps of generating first femtosecond laser pulses by a femtosecond laser source and focusing the first femtosecond laser pulses on a surface or an interior of the photosensitive glass substrate by a focus lens to define a modified region; generating second femtosecond laser pulses by the femtosecond laser source, adjusting a frequency of the second femtosecond laser pulses to be higher than that of the first femtosecond laser pulses by a frequency adjustment unit and an energy adjustment unit; focusing the adjusted second femtosecond laser pulses on the modified region of the photosensitive glass substrate to crystallize a substance of the modified region; and, after crystallization, etching off the crystallized region to obtain the microstructure in the photosensitive glass substrate.