The reductive desorption of a self-assembled monolayer (SAM) of a fluorescent thiol molecule (BodipyC10SH) from Au was characterized using electrochemistry and epi-fluorescence microscopy. Molecular luminescence is quenched near a metal surface, so fluorescence was only observed for molecules reductively desorbed and then separated from the electrode surface. Fluorescence imaging showed that reductive desorption was selective, with desorption occurring from different regions of the Au electrode depending on the extent of the negative potential excursion. When desorbed, the molecules were sufficiently mobile, diffusing away from the electrode surface, thereby preventing oxidative readsorption. At sufficiently negative desorption potentials, all of the thiol was desorbed from the electrode surface, resulting in fluorescence at the air/solution interface. The selective removal of the thiol monolayer from distinct regions was correlated to features on the electrode surface and was explained through potential-dependent interfacial energies. This in situ electrofluorescence microscopy technique may be useful in sensor development.