Indigo, as a blue pigment, has been known as one of the oldest natural dyes. Generally, natural dyes have moderate colors and are environmentally friendly, while harmful mordants must be used for fixing them and obtaining a full color range on fabrics and it is very hard to improve K/S of the dyeing and mordanting. In this study, polygonumtinctroium dye with nano size morphology was prepared using the dorr agitator, homogenizer, and lyophilization and physicochemical characterization of polygoum tinctoria dye substance was investigated by various methods. Then, polygoum tinctoria dyeing was carried out various fabrics according to the various dyeing conditions and the values of K/S according to dyeing conditions were investigated by colorimeter. Also we investigated to dye fastness such as the light, washing, perspiration and friction, cytotoxicity, and antibacterial activity.