Development of sensitive, selective, and reliable assay methods for the detection of pathologically related proteins, especially tumor markers, is a longstanding issue in biomedical research. Herein, we report a sensitive method to detect protein by using peptide-based nano-label fabricated by attaching the streptavidin-specific peptide to streptavidin-coated gold nanoparticle. In the meantime, the nano-label is used in combination with a capture probe prepared by using the specific peptide of the target protein and biotin. In the detection procedure, the target proteins can specifically bind with the biotinylated capture probes which have been previously immobilized on an electrode surface, thus the probes can be protected from thermolysin cleavage. Consequently, the capture probes can be tethered with the nano-labels through the robust biotin/streptavidin interaction, resulting in facile electron transfer between the nano-labels and the electrode. Taking the detection of human glypican-3 (GPC3), a valuable biomarker for hepatocellular carcinoma (HCC),1 as an example, the proposed method can show excellent performance. Moreover, based on the serum level of GPC3 detected by using our method, HCC can be efficiently differentiated from benign hepatic disorders. Owing to its analytical merits and acceptable application in real samples, the proposed method may hold great potential in clinical practice in the future.