The recovery of surfactin from fermentation broths with the culture of Bacillus subtilis ATCC 21332 by physical and chemical extraction was studied, in which the broths were pretreated by acid precipitation and, if necessary, the precipitate was further dissolved in NaOH solution. The physical solid–liquid and liquid–liquid extractions were performed with different organic solvents (ethyl acetate, n-hexane) and at different times of extraction run. It was shown that better extraction was obtained with ethyl acetate than n-hexane. The extraction could be improved by increasing the times of extraction run under a given volume of the organic solution. For chemical liquid–liquid extraction of surfactin with Aliquat 336 (5–200 mM) in n-hexane, the efficiency was improved. The amounts of inorganic salt in the strip solution were also optimized. Surfactin recovered after physical or chemical extraction was finally characterized by mass and NMR spectroscopies. The results of chemical extraction showed that surfactin would readily bind with the quaternary ammonium cations of Aliquat 336.