Abstract: We present an accurate method to determine the effective refractive index and thickness of biomolecular layer by using Fano resonance modes in dual-period gold nanogrid arrays. The effective refractive index changes along the x and y directions are simultaneously measured and obtained by using a modified dispersion relation. The thickness of the surface layer is calculated by a three-layer waveguide equation without any fitting parameters. The accuracy of the proposed method is verified by comparing the results with the known coated dielectric layer and self-assembly layers. The applications of this method and nanogrid chips for determining the thickness and surface concentration of antigen/antibody interactions are demonstrated.