Graphene oxide (GO), an oxidized derivative of graphene, has received much attention for developing novel fluorescent bioanalytic platforms due to its remarkable optical properties and biocompatibility. The reliable performance and robustness of GO-based biosensors have enabled various applications in the biomedical field including diagnosis and drug discovery. Here, recent advances in the development of GO-based fluorescent biosensors are overviewed, particularly nucleic acid detection and enzyme activity assay. In addition, practical applications in biomarker detection and high-throughput screening are also examined. Lastly, basic design principles and remaining challenges of these types of biosensors are discussed for further progress.