Abstract Gold nanoparticles (AuNPs) represent an attractive inorganic matrix for laser desorption/ionization mass spectrometry (LDI-MS) detection of low-molecular-weight analytes; however, their direct use is hindered by severe aggregation. To limit AuNPs aggregation, hexagonal boron nitride nanosheets (h-BNNs) were employed as supports to improve their desorption/ionization efficiency. Thus, Au@BN was synthesized and systematically characterized. It showed low background noise and high sensitivity for LDI-MS of fipronil and its metabolites. Au@BN-assisted LDI-MS was validated using complex samples including blueberry juice, green tea beverage, and fish muscle, achieving low detection limits (0.05–0.20 µg·L−1 for liquid media, 0.82–1.25 ng·g−1 for fish muscle), wide linear ranges (0.2–100 µg·L−1 for liquid media, 3.00–1000 ng·g−1 for fish muscle), high reproducibility (7.55%–13.7%), and satisfactory recoveries (82.62%–109.1%). Furthermore, spatial distributions of analytes in strawberries and zebrafish were successfully imaged. This strategy allows for the quantitative analysis of other small molecules in complex substrates.