We have developed an on-line screening method for CC chemokine receptor 4 (CCR4) ligands, in which the whole cells expressed with CCR4 were cultured adherently and immobilized on the inner wall of the capillary as the stationary phase for the first time. Moreover, in this method it is unnecessary to isolate and purify the target receptors from cell membranes. Therefore, it is possible to almost completely preserve the native conformation of the target receptors. The binding activities of the immobilized CCR4 did not change. A known antagonist of CCR4, compound A, was employed to validate the bioactivity of the cell layer and stability of this method. The intraday, interday, and batch-to-batch reproducibilities were investigated (RSD 13.9%). Nonlinear chromatography was used to calculate the binding constant between the compound A and CCR4 (6.4 x 104/M, RSD = 4.96%). Using this method, the qualitative and quantitative characterizations of 23 computer-aided drug design compounds were achieved and the kinetic parameters (K, ka, kd, and k) were obtained by nonlinear chromatography. Three active compounds were screened out, which also showed activity in chemotaxis inhibition assay. The experimental results show that this method is simple, sensitive, and efficient for drug screening. Moreover, it offered a novel way to detect the nonspecific interactions between ligands and cell membrane.