To investigate the extracted process of gossypol acetic acid (G‐AA) from cottonseed soapstock and explore the improvement of its yield and purity, a novel ultrasound‐assisted extraction and crystallization method was introduced to this process. Under the optimized conditions, preliminary G‐AA with the yield of 1300 mg and the purity of 95.9% could be obtained from 100 g of fresh soapstock by ultrasound‐assisted extraction. In addition, UV, IR, and NMR spectrum further confirmed the detailed chemical structure of G‐AA. Assay of inhibiting human prostate tumor cell line PC‐3 and human breast cancer cell line MDA‐MB‐231 revealed its biological activity, the values of IC50 are 9.096 μmol/L and 14.37 μmol/L respectively. In comparison with the conventional solvent extraction, this novel process increases the content of G‐AA over 90%, reduces the time of crystallization by 75%, and retains the anticancer activity of gossypol. © 2009 American Institute of Chemical Engineers AIChE J, 2009