Surface plasmon enhanced evanescent field at a (noble) metal/dielectric interface can be employed to enhance the diffraction efficiency of surface grating structure composed of biomolecules. Based on a Kretschmann configuration, we realized a diffraction biosensor to monitor the dynamic interaction of biological molecules in a label-free way. It was demonstrated by the binding of an anti-biotin antibody to the biotin-functionalized region of a periodically patterned surface, which generated significant optical contrast to diffract the surface plasmon field. With the aid of the synchronic surface plasmon resonance signal, a quadratic dependence of diffraction signal on the amount of bound antibody was found, which coincides with the theoretical expectation. Time-dependent measurements were conducted to estimate the density of biotin thiols on the functional region.