Diagnosis of apoptosis is essential to the early detection of therapy efficiency and the evaluation of disease progression. Caspase-3 is supposed to be closely related to cellular apoptosis. We describe here a label-free surface plasmon resonance (SPR) detection of apoptosis based on caspase-3 activity assay through enzyme digestion. An artificial peptide sequence was designed as a substrate of caspase-3 and immobilized on a gold disk through covalent binding. The 4Lys part at the end of the pentadecyl-peptide was designed to form a unique peptide array through electrostatic repulsion. The immobilization of the peptide on the gold surface was carefully characterized by SPR and atomic force microscopy. The catalytic conditions of caspase-3 were optimized with electrochemical impedance spectroscopy. The detection limit of caspase-3 was found at a concentration of 1 pg mL(-1). The activity of caspase-3 in apoptotic cells could also be measured sensitively by the one-step and intuitional SPR response decrease. The fabricated simple and convenient caspase-3 sensor is proposed for application in clinical analysis.