We report a novel MoS₂-based fluorescent biosensor for DNA detection via hybridization chain reactions (HCRs). As an emerging nanomaterial, MoS₂ has excellent fluorescence quenching ability and distinct adsorption properties toward single- and double-stranded DNA. In the sensing method, MoS₂ nanosheets are used to suppress the background signal and control the “on” and “off” of fluorescence emission of the detection system with and without the presence of the target DNA. In addition, the signal generation is amplified through the target-triggered HCRs between two hairpin probes. The employment of MoS₂ and HCRs guarantees the high sensitivity of the detection strategy with a detection limit of 15 pM. The biosensor also exhibits very good selectivity over mismatched DNA sequences. The detection takes place in solutions and requires only one “mix-and-detect” step. The high sensitivity, selectivity, and operational simplicity demonstrate that MoS₂ can be a promising nanomaterial for versatile biosensing.