As a soft separation technology with bubble mass transfer, solvent sublation can replace solvent extraction because of many advantages. In this study, solvent sublation was used to separate and purify Penicillin G from fermentation broth. The effects of pH of the solution, NaCl concentration, concentration of butyl acetate (BA) in organic phase, nitrogen flow rate and sublation time on the separation efficiency of Penicillin G were investigated in details, and the optimal conditions for solvent sublation were obtained. The flotation product, which was separated and purified from the fermentation broth under optimal conditions, was quantitatively analyzed by HPLC. Compared with traditional solvent extraction, solvent sublation can not only increase the separation efficiency, but also efficiently reduce the BA emulsification in the separation process. (C) 2008 Elsevier B.V. All rights reserved.