In this study, dyeing, fastness and adsorption isotherm characteristics of madder natural dye on the wool and nylon yarns were investigated. Wool and nylon were first mordanted by aluminum sulfate, dyed with natural dye extracted from madder root, and the rates of dyeing and equilibrium times were determined and compared. Nylon, indeed, showed higher dyeing rate and reached equilibrium at comparatively lower times. Adsorption isotherms were identified to be a Nernst-type substrate. In addition, the affinity (Δµ°), free energy (ΔH°) and entropy (ΔS°) of madder on the wool and nylon were calculated. The obtained results from thermodynamic data indicated that with temperature rise, the partition ratio and the standard affinity significantly decreased. The affinity of madder natural dye to the nylon was gradually higher as compared with wool. Wool, indeed, displayed higher negative values for both enthalpy and entropy changes as compared to those values of nylon. The colorimetric and fastness properties of madder natural dye on nylon were overall superior than on wool.