Serum alpha fetoprotein (AFP) is a "gold-standard" biomarker for the diagnosis of hepatocellular carcinoma (HCC). Available pieces of evidence suggest that the ratio of AFP-L3 isoform in the total AFP may provide more accurate prediction for the incidence of HCC. In this work, we design an electrochemical aptasensor for high-accuracy assay of AFP-L3 ratio based on differentiated labeling of AFP isoforms in an orderly fashion. Specifically, total AFP is first captured by an AFP aptamer-functionalized electrode and labeled with quantum dots-functionalized DNA probes via mild reduction. Then, AFP-L3 isoform that strongly binds to Lens culinaris agglutinin is labeled with silver nanoparticles after the exonuclease-catalyzed removal of DNA probes. By tracing the electrochemical responses of quantum dots and silver nanoparticles, respectively, the amounts of total AFP and AFP-L3 isoforms are determined and the AFP-L3 ratio is accordingly calculated to favor the accurate HCC diagnosis. Experimental results prove the high-accuracy assay of AFP-L3 ratio based on the AFP quantitation in a linear range of 0.0008-40 ng mL -1 and AFP-L3 quantitation in a linear range of 0.004-40 ng mL -1 . The aptasensor also displays satisfactory specificity and good recoveries even in the complex serum samples. Therefore, the aptasensor may provide a valuable tool for the assay of the AFP-L3 ratio and have a great potential use in early warning of HCC for clinical application.