In this paper, we propose a new fluorescence emission difference microscopy (FED) technique based on polarization modulation. An electro-optical modulator (EOM) is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency, which leads to solid- and donut-shaped beams after spatial light modulation. Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve ～λ∕4 spatial resolution. Using the proposed system, the dynamic imaging of subcellular structures in living cells over time is achieved.In this paper, we propose a new fluorescence emission difference microscopy (FED) technique based on polarization modulation. An electro-optical modulator (EOM) is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency, which leads to solid- and donut-shaped beams after spatial light modulation. Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve ～λ∕4 spatial resolution. Using the proposed system, the dynamic imaging of subcellular structures in living cells over time is achieved.