We propose a novel type of immunosensor based on the diffraction of surface plasmon, which allows for in situ, real-time, and label-free observation of interfacial binding events. The inherent self-referencing mechanism of surface diffraction was found to be very effective for compensating fluctuations of the bulk, demonstrated by a temperature variation experiment. Possessing a stable baseline signal, the diffraction sensor offered picomolar sensitivity in directly detecting the binding of human chorionic gonadotropin (hCG) hormone.