As light-emitting nanocrystals, quantum dots (QDs) have created a new realm of bioscience by combining nanomaterials with biology. They also have been a major focus of research and development during the past decade, which will profoundly influence future biological as well as biomedical research. In recent years, near-infrared (NIR) quantum dots have emerged in analytical applications, especially for in vitro and in vivo imaging. The impetus behind such endeavors can be attributed to their unique optical and chemical properties, with size-tunable light emission, high photo stability, and manifold fluorescence colors. In this review, we focus on fluorescent imaging with near-infrared (NIR) quantum dots (QDs) both in vitro and in vivo, and the advantages of QDs and potential problems to their use in practical biomedical applications. The ultimate targets aim at decreasing the cytotoxicity of QDs and the future outlook of QD applications in biomedical fields.