Centrifugal precipitation chromatography and a high-speed counter-current chromatography system equipped with a spiral tubing support rotor (spHSCCC) were successfully applied for the identification and isolation of carotenoid cleavage-like enzymes from Enteromorpha compressa (L.) Nees. This is the first study separating active enzymes from a complex natural matrix by spHSCCC. The target enzymes were identified after fractionation of the proteins in an acetone Tris-buffer gradient by centrifugal precipitation chromatography. Also, an aqueous two-phase solvent system consisting of PEG 1000 and mono- and dibasic potassium phosphate was used for the isolation of the enzymes by spHSCCC. The purified fractions contained two proteins of 65 and 72 kDa, respectively. The enzymes could cleave β-carotene and β-apo-8'-carotenal to produce β-ionone.