This study has developed a surface plasmon resonance (SPR) biosensor with a metal nanoslit structure which couples incident light for real-time analysis of biomolecular interactions. The main advantages of the proposed SPR biosensor are to avoid the disturbance from buffer solution and only need a compact system compared with SPR biosensors with nanostructures. The configuration of the SPR biosensor mainly consists of glass substrate, a metal layer with nanoslits, a biomolecular interaction layer, and buffer solution, sequentially. The incident light first excites the surface plasmons (SPs) which are oscillated on the interface between the glass substrate and the metal layer, and then the emission field of the SPs is coupled via the nanoslits and excites the SPs on the sensing region which is between the metal layer and the buffer solution. The variations of the sensing SPR reflection spectra can be easily detected according to the dynamic biomolecular interaction on the sensing region. The theoretical simulations and the experimental results confirm that the proposed biosensor not only retains the sensitivity based on a grating-coupling SPR biosensor, but also avoids the disturbance from the buffer solution.