A novel three-liquid-phase extraction system (TES) composed of butyl acetate, block copolymer polyethylene oxide-polypropylene oxide-polyethylene oxide and ammonium sulphate aqueous solution [(NH4)2SO4] as top, middle, and bottom phase, respectively, has been developed. The copolymer recycling and partitioning behavior of penicillin V has been studied in this system. Results show that the copolymer could be purified and recycled and penicillin V of the filtrated ferment broth could be partitioned unevenly among the phases and purified in the top phase of this TES. About 90 wt.% of penicillin V could be distributed into the top phase around pH 2.5 and only less than 0.1 wt.% left in the bottom phase.