This technical note reports a method for measuring the diffusion coefficient of molecules based on an optoelectrofluidic platform. Optoelectrofluidic fluorescence microscopy, which is constructed with an optoelectrofluidic device and a conventional fluorescence microscope, is a useful tool for controlling and detecting local molecular concentration in a fluid with a single light source. When we project a light for fluorescence excitation and apply an ac signal of a few hundred Hertz frequency around 100 Hz to the optoelectrofluidic device, a sudden decay of molecular concentration occurs within the illuminated area due to several mechanisms, including optically induced ac electroosmosis, electrostatic interaction forces among the polarized molecules, and interactions among the molecules and between the molecules and the electric double layer. After the applied voltage was turned off, the dispersed molecules diffuse into the molecular depletion area and the fluorescence signal is recovered. On the basis of these phenomena, we successfully measured the diffusion coefficient of various dextran molecules. A statistical analysis to determine the significance of our experimental values compared to the previously reported values measured using fluorescence recovery after photobleaching technology was also performed. This new technique demonstrates the first analytical measurement of diffusion based on the optoelectrofluidic platform and can be a useful tool for measuring the mobility of molecules in a simple and easy way.