Separation and recovery of surfactin from fermentation broths with the culture of Bacillus subtilis ATCC 21332 by two-stage ultrafiltration (UF) or nanofiltration (NF) processes was studied, in which the broths were pre-treated by acid precipitation and then the precipitate was dissolved in NaOH at pH 11. Experiments were performed at different initial concentrations of surfactin (210–3620 mg/L), concentrations of added micelle-destabilizing solvent ethanol (0–44%, v/v), membrane molecular-weight cut-off (MWCO, 1–300 kDa), and transmembrane pressures (86.184–517.106 × 103 Pa (12.5–75 psi)). Under the concentration ranges tested, surfactin micelles could be efficiently destroyed when more than 33% (v/v) ethanol was added to the broth. The UF membranes with MWCO less than 100 kDa were found to be suitable for the retention of surfactin micelles, and the NF membrane with a MWCO less than 1 kDa was suitable for the retention of surfactin monomers. Finally, the separation strategy involving two-stage membrane filtration (UF or NF) processes was proposed. Depending on the forms of the final product, it was shown that more than 72% of recovery yield and more than 83% of purity could be achieved.