Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), are generally composed of II-VI and III-V elements. Due to their quantum confinement of charge carriers in tiny spaces, QDs show some unique and fascinating optical properties, and are characterized as sharp and symmetrical emission spectra, high quantum yields, broad absorption spectra, good chemical and photo-stability and size dependent emission wavelength tunability. Recently, QDs have been successfully used as new fluorescent tags in many biological and biomedical fields, and will become a new promising tool in biomedical studies, clinical diagnostics, drug delivery and photodynamic therapy. In this review, firstly, the methodology of QDs preparation was introduced, which included organic synthesis, aqueous synthesis and microwave assisted aqueous synthesis. Secondly, some procedures for the QDs bio-conjugation with biomarkers were described. And then, some key applications of QDs were summarized, which mainly covered biomedical imaging, immunoassay, DNA hybridization, and photodynamic therapy. Finally, future prospects were discussed.