Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. Recently, the bonding of enzyme to SAMs of alkanethiols onto Au electrode surfaces was exploited to produce a bio-sensing system. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agent. Experimental results have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s core levels at 286.3 and 286.5 eV (C with N), 288.1 eV (amide bond), and 289.3 eV (carboxyl) illustrate the immobilization of trypsin. These data were also in good agreement with Fourier-Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks valued at 1659.4 cm(-1) (amide I) and 1546.6 cm(-1) (amide II). Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) observations, analytical results have demonstrated the platelet proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, platelet proteins were digested on the trypsin-immobilized SAMs surface, which shows the enzyme digestion ability of the immobilized trypsin. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.