In this work, we demonstrate that the analysis of spatially resolved nanofluidic-embedded biosensors permits the fast and direct discrimination of single-nucleotide difference (SND) within oligonucleotide sequences in a single step interaction. We design a sensor with a linear dimension much larger than the channel depth in order to ensure that the reaction over the whole sensor is limited by the convection rate. Thus, the targets are fully collected, inducing a nonuniform spatial hybridization profile. We also use the nanoscale height of the channel, which enables us to minimize the amount of labeled molecules flowing over the sensor and hence to reduce the fluorescence background, to carry out real-time hybridization detection by fluorescence microscopy. Taken together, these design rules allow us to show that the spatial hybridization profile depends on the duplex affinity, and we speculate that the on and off-rate constants can be inferred during target injection, which is not possible in local analysis where the dissociation step through rinsing must be conducted. We finally manage to discriminate a GT mismatch on a microRNA sequence by optimizing the interaction temperature and the probe design after a few minutes of interaction in a single step protocol. This work may be applied to any biosensing transduction scheme with spatial resolution, e.g., surface plasmon resonance imaging, integrated into nanofluidic channels for applications where high oligonucleotide sequence selectivity and short analysis times are required.