Fluorescence microscopy uniquely enables physical and biological research in micro- and nanofluidic systems. However, in channels with depths below 10 nm, the limited number of fluorophores results in fluorescence intensity below the detection limit of optical microscopes. To overcome this barrier, we applied Fabry-Pérot interference to enhance fluorescence intensity with a silicon nitride layer below the sub-10 nm channel. A silicon nitride layer of suitable thickness can selectively enhance both absorption and emission wavelengths, leading to a fluorescent signal that is enhanced 20-fold and readily imaged with traditional microscopes. To demonstrate this method, we studied the mass transport of a binary solution of ethanol and Rhodamin B in 8 nm nanochannels. The large molecular size of Rhodamin B (∼1.8 nm) relative to the channel depth results in both separation and reduced diffusivity, deviating from behavior at larger scales. This method extends the widely available suite of fluorescence analysis tools and infrastructure to unprecedented sub-10 nm scale with relevance to a wide variety of biomolecular interactions.