Glypican-1 (GPC-1) has been recognized as biomarker of pancreatic cancer. Quantification of GPC-1 level is also pivotal to breast cancer and prostate cancer’s patients. We hereby report the first biosensor for GPC-1 detection. Instead of using crosslinking technique and surface immobilization of antibody, we applied a novel method for biosensor fabrication, using S-Acetylmercaptosuccinic anhydride (SAMSA) to modify the Anti-GPC-1 producing a thiol-linked Anti-GPC-1. The thiol-linked Anti-GPC-1 was then directly formed a single-layer antibody layer on the gold biosensor, minimizing the biosensor preparation steps significantly. Time of Flight Secondary Ions Mass Spectroscopy (TOF-SIMS) characterization verified the thiol-linked antibody layer and demonstrated a unique perspective for surface protein characterization. Differential pulse voltammetry (DPV) was applied to quantify GPC-1 antigen in undiluted human serum with a concentration range of 5,000 pg/µL to 100 pg/µL. The performance of this newly designed biosensor was also compared with modified self-assembled monolayer system fabricated biosensor, demonstrating the high-sensitivity and high-reproducibility of the SAMSA modified antibody based biosensor. This simple fabrication method can also expand to detection of other biomolecules. The simplified operation process shows great potential in clinical application development.