Semiconductor quantum dots (QDs), as promising fluorescent materials, have been widely applied in biomedical application due to their unique optical properties. Currently, the most intensively studied are Cd-containing QDs (Cd-based QDs), whose potential toxicity prevents their further commercialization. In recent years, the eco-friendly QDs with low toxicity and environmental friendliness have begun to be developed, showing great potential in biomedical applications. The high-quality synthesis of eco-friendly QDs and the appropriate surface modification are key to realize their applications. This review summarizes the progress of eco-friendly QDs for biomedical applications, including their designed preparation, optical properties, surface modification, toxicity, and their applications in bioimaging and diagnostics. Finally, the challenges of eco-friendly QDs for future bioimaging and diagnostics application were provided. We believe this review will provide important guidance for promoting the development of eco-friendly QDs in bioimaging and diagnostics.