Indigo, as a blue pigment, has been known as one of the oldest natural dyes. Generally, natural dyes have moderate colors and are enviromentally friendly, but harmful mordants must be used for fixing them and obtaining a full color range no fabric and it is very hard to improve K/S values of the dyeings and mordanting. In this study was investigated physicochemical characterization of polygoum tinctoria dye substance to solve these problem. Calcium hydroxide was added into polygoum tinctoria leaves to precipitate dye substance and it was freezing dried into powder form. It's characterization was investigated to UV/VIS spectrophotometer, TLC and DSC. Indigo and Indirubin was separated from polygoum tinctoria powder for soxhlet extraction, and these structure and characterization were investigated to FT-IR spectrum.