Polyvinyl alcohol (PVA), a synthetic polymer produced by polymerizing vinyl acetate and subjecting it to alkaline hydrolysis, exhibits properties such as film formation, emulsification, and adhesion. However, pure PVA’s biocompatibility and mechanical properties are limited. Its hydrophilicity also causes it to dissolve in water and blood, rendering it less suitable for drug delivery applications. To address these limitations, PVA was crosslinked with glutaraldehyde (GA) to enhance toughness and resistance to water and blood dissolution. Additionally, carboxyl (COOH) and hydroxyl (OH) functionalized graphene were incorporated into electrospun PVA nanocomposites to further improve their mechanical properties. The results show that adding COOH- and OH-functionalized graphene in four different concentrations (0.5, 1.0, 1.5, and 2.0 wt.%) significantly enhanced the mechanical characteristics of PVA nanocomposites. Tensile strength and Young's modulus increased substantially, with OH-functionalized graphene increasing tensile strength and Young's modulus by 224% and 338.3%, respectively, and COOH-functionalized graphene increasing these properties by 245.6% and 371.4%. Morphological characterization using FT-IR and FESEM confirmed the successful incorporation of graphene into the PVA matrix. Biocompatibility testing through APTT and PT assays showed both nanocomposites are biocompatible, suggesting their potential for biomedical applications. The optimal filler concentration for both graphene types was 1.5 wt.%. This research demonstrates the promising potential of innovative materials for healthcare and biomedical engineering applications.