A novel method to improve the axial resolution of fluorescent microscopy using photo-switching characteristics of fluorescent protein is proposed. For microscopy with different numerical apertures, a 100-nm axially-selective-activation is achieved by using cylindrical vector beams with vortex phase as activation and deactivation beams which are modulated by designed diffractive optical elements. Furthermore, adjusting the power ratio R of the activation beam and deactivation beam will make the maximum activation probability of fluorescent protein suitable for single-molecule microscopy. The numerical calculation based on Cy5 demonstrates that along the z axis the activation probability has a single peak with full-width at half-maximum (FWHM) of only 25.7 nm and about 90% of activated molecules are located in a 30-nm-thick layer around zero point (for R=500).