Hexagonal boron nitride (hBN) is gaining increasing attention in the field of biomolecule characterization due to its compatibility with single-molecule fluorescence imaging and real-time tracking. Embedding fluorescent molecules within hBN layers offers potential for molecular-resolution sensing devices, since these probes are highly sensitive to their surroundings. Yet, the effect of hBN surfaces on the fluorophore properties remains largely unexplored. Here, we monitor the photophysical properties of ATTO647N-ssDNA on hBN surfaces and elucidate the effects of the environment and substrate. We demonstrate that the presence of hBN increases the photobleaching time and changes intermittency dynamics. By combining van der Waals stacking and FDTD simulations, we subsequently engineer hBN optical cavities to modulate the emission from individual molecules, showing that the brightness can be tuned by a factor of 4. Our findings shed light on light–matter interactions in hybrid nanostructures, which can enable single-molecule imaging and biosensing at high spatial and temporal resolution.