Graphene quantum dots (GQDs) have generated enormous excitement because of their superiority in chemical inertness, biocompatibility and low toxicity. Due to quantum confinement and edge effects, GQDs have excellent properties, attracting extensive attention from scientists in the fields of chemistry, physics, materials science, biology, and other interdisciplinary sciences. In this review, we aim to present a comprehensive view on the synthesis of GQDs for biological applications. We highlight potential methods like acid oxidation, hydrothermal and solvothermal reactions, microwave-assisted methods, electrochemical oxidation, as well as pyrolysis and carbonization for the successful preparation of GQDs. Meanwhile, four representative types of biomedical application based on GQDs, bioimaging, biosensing, drug delivery, and antimicrobial materials, are introduced and discussed in detail. This work will be very useful for quickly gaining knowledge and experience for synthesizing various GQDs, and developing advanced strategies for creating novel functional GQD-based nanomaterials for further applications in biomedicine, materials science, analytical science, and optical nanodevices.