An aqueous two-phase system (ATPS) was presented with hydrophilic ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) and NaH2PO4 aqueous solution in this paper. The partitioning behavior of penicillin G in the ATPS was investigated. Concentrations of NaH2PO4, penicillin G, and [Bmim]Cl were evaluated to determine their effects on the partition coefficient and extraction yield of penicillin G. It was found that both of partition coefficient and extraction yield strongly depended on the concentration of [Bmim]Cl, penicillin and NaH2PO4. A high extraction yield of 93% was achieved with the following parameters: NaH2PO4 center dot 2H(2)O 40% (wt%), penicillin 45000 similar to 50000 u/ml, [Bmim]Cl 20 similar to 21% (wt%). The [Bmim]Cl/NaH2PO4 system was also applied in a real filtration of penicillin G fermentation broth and the extraction yield was averaged at 91.5%. It is worthy noting that the working pH value of ATPS was at the range of 5 similar to 6, no emulsification and protein denaturation could be observed.