The invention discloses a method for two-photon fluorescence stimulated emission differential super-resolution microscopy. The method includes the steps that (1) after being collimated, pulsed laser beams are converted into linear polarized light and polarization modulation is conducted on the linear polarized light, so that radial polarized light is obtained; (2) the radial polarized light is converted into circular polarized light and projected onto a sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore first signal light intensity I1 is obtained; (3) polarization modulation is conducted on the linear polarized light obtained in the step (1) and the linear polarized light is converted into tangential polarized light; (4) the tangential polarized light is converted into circular polarized light and projected onto the sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore second signal light intensity I2 is obtained; (5) effective signal light intensity I is calculated according to a formula I=I1-gammaI2 , so that super-resolution imaging is achieved. The invention further discloses a device for two-photon fluorescence stimulated emission differential super-resolution microscopy. The device is simple, free of light division, low in light power, capable of weakening the photobleaching effect, higher in resolution and larger in imaging depth.