<p>Graphene oxide, a 2D nanomaterial, has attracted significant attention for biomedical applications due to its exceptional properties, including tunable surface chemistry, excellent dispersibility, and abundant oxygen-containing functional groups that facilitate facile modification. Recent advancements in the synthesis and functionalization of graphene oxide-based materials have addressed their limitations, such as poor solubility and cytotoxicity, thereby making them safer and more effective for biomedical applications. This paper thoroughly outlines the structural attributes and principal qualities of graphene oxide-based materials, highlighting their mechanical strength, thermal stability, impermeability, electrical conductivity, and biological properties. Emerging developments in graphene oxide-based nanocomposites for fluorescence imaging, magnetic resonance imaging, photoacoustic imaging, Raman spectroscopy imaging, and multifunctional therapeutic platforms are thoroughly examined. Their effectiveness in biomedical scaffolds, wound-healing systems, controlled drug-release platforms, and antimicrobial coatings has been highlighted. Notwithstanding considerable advancements, issues related to cytotoxicity, biodegradability, long-term biosafety, and scalable production continue to impede broad clinical translation. This study offers a cutting-edge overview of graphene oxide-based biomedical systems and outlines promising pathways for advancing safer, more effective, and clinically relevant graphene-based healthcare technologies.</p>