Abstract: The aim is to get the optimum determination wavelength of reducing sugar quantitatively determined by DNS method. The samples were scanned by UV visible spectrophotometer, and then the absorbance and the concentration of reducing sugar were linearly fitted and analyzed at each wavelength. And to verify whether the wavelength selection is reasonable, the RSD of 10-time scan results of the same sample was analyzed. The results showed that the linear correlation coefficient (R2) of glucose was very close to R2 max at 520 nm ( R2 520 ≈ R2 max ). In the range of 520-580 nm, R2 was stable at 0.9998 and the slope decreases with the increase of wavelength. R2 of galactose was also very close to the R2 max at 520 nm ( R2 520 ≈ R2 max ). In the range of 520- 580 nm, R2 and the slope also decreased with the increase of wavelength. The results of RSD analysis of the same sample showed RSD520≈RSDmin. In addition, the absorbance value of control group was stable in the range of 480-580 nm, while the absorbance value of the experimental group fluctuated significantly in the short wavelength range, the smaller the wavelength, the greater the fluctuation. The results showed that the better linear relationship and resolution could be got at 520 nm, which was verified by the verified experiment of the same sample. The absorbance fluctuation of the experimental group showed that it was unreasonable to choose a shorter wavelength; it also showed that the light absorbing group of the newly formed amino compound was different from the original light absorbing group of the DNS.