We propose biofunctionalized nanofluidic slits (nanoslits) as an effective platform for real-time fluorescence-based biosensing in a reaction-limited regime with optimized target capture efficiency. This is achieved by the drastic reduction of the diffusion length, thereby a boosted collision frequency between the target analytes and the sensor, and the size reduction of the sensing element down to the channel height comparable to the depletion layer caused by the reaction. Hybridization experiments conducted in DNA-functionalized nanoslits demonstrate the analyte depletion and the wash-free detection ∼10 times faster compared to the best microfluidic sensing platforms. The signal to background fluorescence ratio is drastically increased at lower target concentrations, in favor of low-copy number analyte analysis. Experimental and simulation results further show that biofunctionalized nanoslits provide a simple means to study reaction kinetics at the single-pixel level using conventional fluorescence microscopy with reduced optical depth.