Quantum dots (QDs) have been facilitating the development of sensitive fluorescence biosensors over the past two decades due to their high quantum yield, narrow and tunable emission spectrum and good photostability. The new emerging QDs with improved biocompatibility further promote their biological applications. In this review, we first briefly introduce the prevalently used QDs and their preparation and bioconjugation approaches. Then we summarize QDs-based fluorescent biosensing for proteins and nucleic acids, and QDs-based applications in cellular and in vivo targeting and imaging. Last but not the least, we envision the potential QDs-based applications in future perspectives.