The separation of penicillin G from water-butanol mixed solution using strongly basic anion resin was investigated. The comparison by between gel-type and macro porous-type resin was also carried out at static and dynamic conditions, and it is demonstrated that the get-type 201×4 resin in Cl form can absorb more penicillin G from the water-butanol mixed solution. The elution results show that the addition of organic solvent into the mixed eluant can make great improvement in the eluant concentration, and hence the suitable elution conditions are obtained. It is indicated that the recovery of penicillin G from water-butanol mixed solution by ion exchange method is feasible.