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Herein we developed a highly sensitive methodology for heparin detection based on a resonance Rayleigh scattering (RRS) spectral assay. Heparin and concanavalin A (conA) densely stacked together and formed nanoparticles or a well-defined brick-and-mortar nanostructure. A strong electrostatic attraction, hydrogen bond and the solid-liquid interface induced RRS enhancement. The result of absorption spectra and TEM testified that the introduction of heparin indeed changed the structure of conA and formed brick-and-mortar nanostructures. The optimum reaction conditions of the method and the reasons for RRS enhancement are also discussed. Combined with an RRS spectral assay, an effective biosensor has been developed for heparin. The bioassay allows sensitive detection of heparin with a detection limit of 2.48 ng mL À1 (3s), a linear range from 8.28 ng mL À1 to 2.5 mg mL À1 . The method was successfully applied to the determination of heparin in heparin sodium injection samples.