A facile and convenient process was optimized for preparing water-soluble hydroxyl-functionalized hexagonal boron nitride (hBN-OH) from hBN. The hBN-OH (2–3 nm thickness) contains ∼40% oxygen and exhibits blue emission with a quantum yield of approximately 36%. The hBN-OH could be used for imaging cells and for the in vitro detection of biomolecules through electrochemical analysis.