Five guanido packings of affinity chromatography for the separation of urokinase have been synthesized. The separation characteristics and the effect of matrix and ligands on them have been studied. The guanidine derivative is a fine ligand, the packings with ligand of arginine are much better than that with ligand of p-ABZ. The polyepoxypropyl methacrylate microsphere is a good matrix. The comprehensive function of packings with ligand of arginine and matrix of polyepoxypropyl methacrylate microsphere is more excellent than Sepharose-p-ABZ.