In this article, a surface plasmon resonance (SPR) biosensor based on graphene oxide (GO) for simple and rapid determination of D-dimer in plasma is introduced. GO was conjugated onto the surface of the SPR chip surface via electrostatic interaction after the chip surface was activated with cysteamine. Subsequently, anti-D-dimer was covalently immobilized on the chip surface through binding between carboxylic groups on the GO surface and amine groups on the protein surface. Finally, label-free and rapid detection of D-dimer was carried out by the SPR biosensor via protein-protein interaction. After optimization, the concentration of GO was found to be 2 mg mL −1 . Meanwhile, this biosensor presented a response to D-dimer in the concentration range of 10-150 ng · mL −1 , and limit of detection (LOD) of the developed biosensor was determined to be as low as 5.08 ng · mL −1 .In addition, addition of albumin from human serum (HSA) to the anti-D-dimer-modified SPR chip revealed a negligible response, indicating a satisfactory immunological specificity. Furthermore, D-dimer detection was successfully conducted in different concentrations of human plasma. These results suggest that the GO-assisted SPR biosensor could be exploited for point-of-care diagnosis in the clinical laboratory in the future.