AbstractInP quantum dots (QDs) are a promising and environment‐friendly alternative to Cd‐based QDs for in vitro diagnostics and bioimaging applications. However, their poor fluorescence and stability severely limit their biological applications. Herein, we synthesize bright (∼100%) and stable InP‐based core/shell QDs by using cost‐effective and low‐toxic phosphorus source, and then aqueous InP QDs are prepared with quantum yield over 80% by shell engineering. The immunoassay of alpha‐fetoprotein can be detected in the widest analytical range of 1–1000 ng ml−1and the limit of detection of 0.58 ng ml−1by using those InP QDs‐based fluorescent probes, making it the best‐performing heavy metal‐free detection reported so far, comparable to state‐of‐the‐art Cd‐QDs‐based probes. Furthermore, the high‐quality aqueous InP QDs exhibit excellent performance in specific labeling of liver cancer cells and in vivo tumor‐targeted imaging of live mice. Overall, the present work demonstrates the great potential of novel high‐quality Cd‐free InP QDs in cancer diagnosis and image‐guided surgery.