Abstract: Based on the ultraviolet (UV) absorption characteristics of glucosamine, the UV detection wavelength of chitooligosaccherides was optimized to 200 nm by detecting the standard solution of glucosamine at the wavelength of 190-400 nm. Two kinds of commercial chromatographic column (Shodex amino column and TOSOH amide column), commonly used in analysis of oligosaccharides, were tested to compare the separation effect of glucosamine, and the results showed that the separation resolution of two columns was similar, but the peak time of the former was earlier. Based on Shodex amino column, the standard curve of glucosamine for quantitative calculation was simulated successfully by UV detector. Lastly, a chromatographic separation method of chitooligosaccharides for qualitative and quantitative detection was verified. It was found that the resolution and the distribution peak of chitooligosaccharides with different degree of polymerization, were similar to those of differential refractive index detector. At the same time, the degree of polymerization distribution of chitooligosaccharides mixed standard sample (DP 2-7) was determined. As a conclusion, the proposed method was a valid supplement for the current standard detection method of chitooligosaccharides industry.