A new type of functional optical microscope system called three-dimensional (3D) stimulation and imaging-based functional optical microscopy (SIFOM) is proposed, to the best of our knowledge. SIFOM can precisely stimulate user-defined targeted biological cells and can simultaneously record the volumetric fluorescence distribution in a single acquisition. Precise and simultaneous stimulation of fluorescent-labeled biological cells is achieved by multiple 3D spots generated by digital holograms displayed on a phase-mode spatial light modulator. Single-shot 3D acquisition of the fluorescence distribution is accomplished by common-path off-axis incoherent digital holographic microscopy in which a diffraction grating with a focusing lens is displayed on another phase-mode spatial light modulator. The effectiveness of the proposed functional microscope system was verified in experiments using fluorescent microbeads and human lung cancer cells located at various defocused positions. The system can be used for manipulating the states of cells in optogenetics.