We report three-dimensional fluorescence emission difference (3D-FED) microscopy using a spatial light modulator (SLM). Zero phase, 0–2π vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid, doughnut and z-axis hollow excitation spot, respectively. Our technique achieves super-resolved image by subtracting three differently acquired images with proper subtractive factors. Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED. Also, the improvement of lateral and axial resolution is demonstrated by imaging 100 nm fluorescent beads. The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.