A new type of 0-dimensional carbon-based materials called graphene quantum dots (GQDs) is gaining significant attention as a non-toxic and eco-friendly nanomaterial. GQDs are nanomaterials composed of sphybridized carbon domains and functional groups, with their lateral size less than 10 nm. The unique and exceptional physical, chemical, and optical properties arising from the combination of graphene structure and quantum confinement effect due to their nano-size make GQDs more intriguing than other nanomaterials. Particularly, the low toxicity and high solubility derived from the carbon core and abundant edge functional groups offer significant advantages for the application of GQDs in the biomedical field. In this review, we summarize various synthetic methods for preparing GQDs and important factors influencing the physical, chemical, optical, and biological properties of GQDs. Furthermore, the recent application of GQDs in the biomedical field, including biosensor, bioimaging, drug delivery, and therapeutics are discussed. Through this, we provide a brief insight on the tremendous potential of GQDs in biomedical applications and the challenges that need to be overcome in the future.