Natural dyestuff of luteolin was isolated and used to dye wool fabric in this paper. Ethanol extraction and high-speed countercurrent chromatography (HSCCC) were used to extract and purify the luteolin from the peanut shell, and the structure of the isolated luteolin was characterized with FTIR techniques. The interaction between dyestuff and fiber was preliminarily discussed through thermodynamic study and supramolecular structure simulation to explain the intrinsic reasons why the color fastness was low when luteolin was applied to dyeing wool fabric. The extraction condition and purification parameter were as follows: 65% ethanol, ratio of material to liquid 1:20, 80˚C, 3 h, chloroform-methanol-water (4/3/2, V/V), 800 rmp/min, 2.0 Mkpa, 0.5 mL/ min and 280 nm. The results of dyeing thermodynamics showed that the sorption isotherm of luteolin on wool fabric was consistent with Nernst model and similar to the disperse dyestuff. With molecular simulation, luteolin and glycin composed 8 stable complexes whose Laplacian values all were greater than 0, which suggested typical hydrogen bonds existing. The complex with three hydrogen bonds was proved the most stable. Both studies on thermodynamics and supramolecular simulation revealed that luteolin on wool fabric mainly depended on the weak hydrogen bonds interaction that determined the low dyefastness.