An extraction method of madder (Rubia tinctorum) roots dyes is established and optimized to obtain the original chemical composition. A central composite design (CCD) was developed to specify the importance of the three major factors studied (time, temperature and solvent composition) affecting the ultrasound-assisted extraction of this matrix. A preliminary granulometric study of madder roots is realized in the aim to determine the optimal particles size corresponding to the best ultrasound effects. A comparison with the classical extraction method of madder dyes by reflux is described. The identification of the constituents of R. tinctorum is carried out by liquid chromatography coupled with a photodiode array detector (LC–PDA). Anthraquinonic aglycone and heterosidic dyes compounds are characterized by retention time and UV spectrum: alizarin (1,2-dihydroxyanthraquinone), purpurin (1,2,4-trihydroxyanthraquinone), lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone), rubiadin (1,3-dihydroxy-2-methylanthraquinone), xanthopurpurin (1,3-dihydroxyanthraquinone), pseudopurpurin (1,2,4-trihydroxy-3-carboxyanthraquinone), lucidin primeveroside, ruberythric acid (alizarin primeveroside), galiosin (pseudopurpurin primeveroside) and rubiadin primeveroside. The optimal experimental conditions are 18 min, 36 °C and 37/63 MeOH/H2O (v/v)