A non-toxic biomimetic interface for immobilization of living cells and electrochemical exogenous effect study of cell viability was constructed by mixing colloidal gold nanoparticles in carbon paste. A new approach to study the effects of anti-tumor drug and other exogenous factors on cell viability was proposed. The nanoparticles were efficient for preserving the activity of immobilized living cells and preventing their leakage from the electrode surface. The immobilized living AsPC-1 cells (pancreatic adenocarcinoma cells derived from ascites) exhibited an irreversible voltammetric response related to the oxidation of guanine. The presence of guanine was verified by liquid chromatography-mass spectrometry. The contents of guanine in cytoplasm of each AsPC-1 and normal pancreatic cell were detected to be 370 and 22amol, respectively. The cytotoxic effect of adriamycin resulted in a decrease in peak current of guanine. The optimal exogenous factors that affected cell viability, including pH, temperature and salt concentration of electrolyte, were just consistent with cell growth conditions in culture. This simple and rapid method could be applied for the electrochemical investigation of exogenous effect and characterization of the viability of living cells.