Seeds and leaves of indigo (Polygonum tinctorium Ait.) plant were investigated and compared with another medicinal plant named prolipid for their properties such as chemical composition, antioxidant, and anticancer effects by Fourier transform infrared, three-dimensional fluorescence spectroscopy, and electrospray ionization-MS in negative mode. It was found that polyphenols, flavonoids, and flavanols were significantly higher in prolipid (P < 0.05), following by indigo mature leaves, immature leaves, and seeds. Methanol extract of mature indigo leaves in comparison with the ethyl acetate extract showed higher inhibition of proliferation. The interaction between polyphenol extracts of indigo mature leaves and BSA showed that indigo has a strong ability, as other widely used medicinal plants, to quench the intrinsic fluorescence of BSA by forming complexes. In conclusion, indigo mature leaves were compared with prolipid. High content of bioactive compounds, antioxidant, fluorescence, and antiproliferative properties of indigo justifies the use of this plant as a medicinal plant and a new source of antioxidants.