In this study, a surface plasmon resonance (SPR) biosensor with sub-wavelength grating waveguide for the real-time analysis of biomolecular interactions is developed. The conventional SPR has diffractive grating structure to increase the wave vector for exciting the surface plasmons and then detects biomolecular interactions in high order diffraction light. Using this approach has some disadvantages such as the intensity of high order diffraction light is dimmer to be difficult to measure and the measured reflectivity spectrum is too broadened. The proposed SPR biosensor uses a normally incident white light with the help of subwavelength grating structure and provides a sharper reflectivity spectrum according to waveguide interference both to avoid disadvantages of the conventional SPR biosensor with a grating coupler. When the diffraction grating waveguide structure and the condition of SPR are destroyed by external factors such as slight refractive index changes of the buffer or molecule adsorption on the grating surface, the optical path and momentum of the light coupled through the gold grating into the waveguide are changed and a resonance wavelength shift is induced as a result. By detecting this resonance wavelength shift, the SPR 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. The proposed biosensing metrology system becomes more simply and convenient for real-time biomolecular interaction analysis.