Abstract: To analyze the relationship of Polygonum multiflorum Thunb slice among processing degree, color and in vitro viability, a rapid non-destructive evaluation for color data was built. Cell viability assay in vitro and total polysaccharide, total anthraquinone were determined by ultraviolet spectrophotometry and MTT assay, and colorimeter to the color values of Polygonum multiflorum Thunb slice was measured. Cluster analysis and spearman analysis were employed to analyze the correlation between samples with different processing degree and the correlation analysis of multiple-factor. Results showed that, with the increasing of processing degree, the color values L^*, a^* and b^* of Polygonum multiflorum Thunb slice were at a downward trend. The processing in the 6^th to 9^th time could significantly promote cell viability. Color difference analysis showed that L^*, a^*, b^* were at 34.38~36.11, 3.44~4.22, 1.45~3.24.And the total color difference E^*ab was 34.58~36.50, while the Polygonum multiflorum Thunb slice had the best effect on cell viability reaching 135.53%~153.14%. The PLS regression model of Polygonum multiflorum Thunb slice about cell viability, colors and polysaccharide was constructed as follows:Cell viability (Y)=0.434×L^*-0.239×a^*+0.317×b^*+0.235×polysaccharide, and the rate of cell viability was high when Y was 14.7~17.2.It is firstly reported the correlation between the color change, chemical components and cell proliferation in the preparation of Polygonum multiflorum Thunb, which forecasts quickly the content of total polysaccharide as well as the contents of proliferation rate in cells, and provides a new method for the establishment of the evaluation system for the Chinese medicine Heshouwu.