In this work, we propose a sensitive method to detect proteins by using a peptide-based nano-label. This label is fabricated by attaching the streptavidin-specific peptide to a 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), as an example, experimental results demonstrate that the proposed method can show excellent performance. Moreover, based on the serum level of GPC3 detected by our method, HCC can be efficiently differentiated from benign hepatic disorders. Owing to its analytical merits and acceptable applications in real samples, the proposed method may hold great potential in clinical practice in the future.