In this paper, the reflection resonance spectrum of a sub-wavelength diffraction grating-coupled waveguide is used to analyze biomolecular interactions in real time. When the diffraction grating waveguide structure is destroyed by external factors such as slight refractive index changes of the buffer or molecule adsorption on the grating surface, the optical path of the light coupled through the grating into the waveguide is changed and a resonance wavelength shift is induced as a result. By detecting this resonance wavelength shift, the optical waveguide biosensor provides the ability to identify the kinetics of the biomolecular interaction on an on-line basis without the need for the extrinsic labeling of the biomolecules. A theoretical analysis of the sub-wavelength optical waveguide biosensor is performed. A biosensor with a narrow reflection resonance spectrum, and hence an enhanced detection resolution, is then designed and fabricated. Currently, the detection limit of the optical waveguide sensor is found to be approximately 10<sup>-5</sup> refractive index units. The developed biosensor is successfully applied to study the kinetics of an antibody interaction with protein G adsorbed on the sensing surface.