Objective: To establish a method for isolating and purifying actin from Dosidicus gigas myofibrillary protein and realize the batch preparation of actin. Method: The mixed protein of myofibrillar was extracted from Dosidicus gigas by phosphate buffer. The protein extracted from the squid was separated by anion exchange chromatography with only a small charge difference. Then the gel filtration chromatography was used to remove the small molecules in the mixture， such as desalination， and then purified by macromolecular protein purification. The purified protein was obtained with high purity (AC). SDS-PAGE， HPLC and MALDI-TOF-MS were used to identify the purity and molecular weight of the isolated protein. The particle size distribution of actin in the solution was characterized by light scattering technique. Results: In this paper， actin with high purity was extracted from myofibrillar protein， and the hydraulic radius(Rh) of actin was 3.46 nm. Conclusion: A new method for isolation and purification of actin in myofibrillar protein was preliminarily established， which provided support for the batch preparation of actin.