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A convenient and sensitive triple-wavelength overlapping resonance Rayleigh scattering (TWO-RRS) method for the detection of chito-oligosaccharides (COS) was proposed based on enhancing the rigid surface of porous reticular spatial structure of gelatin and COS by introducing allura red AC (AR). The interaction and resultant porous reticular spatial structure were characterized with transmission electron microscopy (TEM), RRS, and UV-Vis spectroscopy. The results indicated that gelatin and COS formed porous reticular spatial structure with an average diameter of 1.5–2.0 μm, and the RRS value of COS-AR-gelatin ternary system with gelatin participation was significantly higher than that of COS-AR binary system. Under the optimal conditions, the enhanced TWO-RRS intensity of the system was linearly proportional to COS concentration in the range of 0.30–2.50 μg/mL, and the regression equation was ΔI = 4933.2c − 446.21 with R2 = 0.9980. The limit of detection was 0.0478 μg/mL. So, a new method for the detection of COS was established and verified in the health products with satisfactory results.

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