Abstract Rare-earth doped upconversion nanophosphors (UCNPs), which convert low energy near-infrared (NIR) photons into high energy photons such as ultraviolet, visible light and NIR light, have found various applications in optical bioimaging. In this review article, we summarize recent advances in the synthesis and applications of UCNPs achieved by us and other groups in the past few years. The approaches for the synthesis of UCNPs are presented, with an emphasis on the role of green chemistry in the advancement of this field, followed by a focused overview on their latest applications in optical bioimaging from subcellular structures through cells to living animals. Challenges and opportunities for the use of UCNPs in biomedical diagnosis and therapy are discussed.