In this work, by functionalizing MoS₂ quantum dot with 3-aminobenzeneboronic acid, a novel multifunctional quantum dot (denoted as B-MoS₂ QD) was obtained and used successfully for a fluorescence nanoprobe for detecting o-dihydroxybenzene (o-DHB). Transmission electron microscopy, fluorescence spectrum, UV-vis spectrum and fluorescence lifetime were used to investigate the prepared nanoprobe. The results show that the B-MoS₂ QD nanoprobe can exhibit strong fluorescence and excellent light fastness owing to the coupled effect from the MoS₂ QDs and boronic acid; interestingly, the vicinal diols structure from its surface can bridge covalently with o-DHB, resulting in the fluorescence quenching of B-MoS₂ QDs and selective recognition toward o-DHB. With the increasing of o-DHB concentration, the nanoprobe fluorescence would gradually decrease. By measuring the fluorescence intensity of B-MoS₂ QDs, a wide linear range from 0.1 to 200.0 μM with a low detection limit of 0.025 μM was obtained for o-DHB analysis; meanwhile, this fluorescence nanoprobe possesses excellent selectivity for the selective detection of o-DHB from its analogues.