In this study, we investigate fluorescence optical detection and image reconstruction based on modulated light illumination using a digital micromirror device (DMD). Fluorescence detection is one of the most common methods to study various cellular dynamics labeled with fluorescent indicators. Although employed in many cell-based assays, widefield fluorescence microscopy provides poor axial sectioning capability that is insufficient to measure thick cell-based assays, e.g., those with 3D cell complex cultured in a thick extracellular matrix. Confocal fluorescence microscopy, on the other hand, provides good axial sectioning capability compared to wide-field fluorescence imaging. However, confocal microscopy is subject to temporal overhead associated with scanning to acquire fluorescence images. For image acquisition at enhanced axial sectioning with reduced processing load, we have developed a fluorescence optical detection system based on subtractive light illumination using a DMD. Compared to moving grid masks, a DMD provides fast and flexible scanning by modulating aperture patterns. Here, we report DMD-based structured light illumination for improved image reconstruction by separating in-focus and out-of-focus fluorescence components so that the system can achieve 3D fluorescence image stacks with enhanced axial sectioning capability. In this proof-of-concept study, the DMD-based structured light illumination system was evaluated by observing two-dimensionally deposited fluorescent microbeads and mixed pollen grains. Also shown was that the scanning time to obtain fluorescence images can be greatly reduced compared to confocal microscopy.