We propose a novel fluorescence emission difference microscopy (FED) system based on focusing cylindrical vector beams. In conventional FED, a Gaussian beam and a 0–2π vortex phase plate are used to generate solid and hollow spots. We focus radially polarized and azimuthally polarized cylindrical vector beams to obtain an expanded solid spot and a shrunken hollow spot, taking advantage of the optical properties of cylindrical vector beams to improve the conventional FED performance. Our novel method enhances FED performance because the hollow spot size determines the FED resolution and an expanded solid spot effectively reduces negative side-lobe emergence during image processing. We demonstrate improved performance theoretically and experimentally using an in-house built FED. Our FED achieved resolution of less than λ/4 in test images of 100 nm nanoparticles, better than the confocal image resolution by a factor of approximately 1/3. We also discuss detailed simulation analyses and FED imaging of biological cells.