The natural dye substances appear to have a lot of benefits that enable the dyed materials to exhibit their natural colors. It may have a bit of a harmful influence on the human body itself, in comparison with the synthetic substances. It also causes hardly any environmental pollution because of the natural chemicals and costs much less to disposal. In this study was investigated physicochemical characterization of polygoum tinctoria dye substance and synthetic indigo. Calcium hydroxide was added into polygoum tinctoria leaves to precipitate dye substances and it was freezing dried into powder form. characterizations of dye substances were investigated by UV/VIS spectrophotometer, TLC, DSC, TGA, HPLC, and MALDI-TOF Mass. Resultantly, polygoum tinctoria was confirmed that is consisted of two ingredients from these results.