Medical diagnostics have been expanded to new dimensions by graphene and its derivatives due to their unique chemical and physical characteristics, including excellent electrical and thermal conductivity, a large specific surface area, and easy biofunctionalization combined with low fabrication costs. Thereby, graphene-based materials have been widely used as a promising nanoplatform for nano-scale sensor and biosensor fabrication. Moreover, the molecular structures of graphene-based materials, especially oxygenated functional groups, facilitate their chemical functionalization and enable combining graphene-based nanoparticles with other inorganic and organic nanomaterials, biological polymers, and quantum dots to form a wide range of nanocomposites with improved sensitivity and selectivity for sensor applications. This chapter focuses on the synthesis and characterization of graphene-based nanocomposites for quantitative detection of significant small biomolecules, including uric acid (UA), ascorbic acid (AA), dopamine (DA), and tryptophan (Trp), in human metabolism. It also updates readers with recent advances and scientific progress in using graphene-based nanocomposites in sensing and biosensing applications. Finally, the future prospects of graphene-based biosensor development, along with their challenges and potential answers, are discussed.