This study explored the effects of multiple freeze-thaw cycles on myofibrillar protein intermolecular force of Sepia esculenta and its protein properties. Fresh S. esculenta meat were defrosted by three different ways which are 0℃, running water and normal temperature for 9 freeze-thaw cycles. The results showed that the effects of defrosting ways were significant on intermolecular forces, and thaw at 0℃ is better than running water and normal temperature. With the increase of freeze-thaw times, the content of ionic bond and hydrogen bond showed a significant decline, the ionic bond decreased by 7.71%, 10.64% and 13.61%, respectively, and the hydrogen bond decreased from 31.91%, 31.97% and 31.87% to 26.76%, 25.53% and 23.94%. On the contrary the hydrophobic interaction force, disulfide bond and non-disulfide covalent bond tended to increase, the hydrophobic forces increased by 8.86%, 12.35%, and 14.72%, respectively, and the disulfide bonds increased by 1.43%, 1.96%, and 2.85%, respectively, the non-disulfide covalent bonds increased from 1.16%, 1.28%, and 1.55% to 3.75%, 4.05% and 5.50%. The surface hydrophobicity of myofibrillar protein increased from 30.47 μg, 31.31 μg and 32.26 μg to 46.10 μg, 53.51 μg and 58.91 μg, respectively, consistent with hydrophobic force results. Both sulfhydryl and reactive thiol content were decreased, the sulfhydryl groups were reduced by 13.07 nmol/mg, 38.99 nmol/mg and 40.32 nmol/mg, respectively, and the active sulfhydryl groups were decreased by 6.55 nmol/mg, 24.26 nmol/mg and 43.16 nmol/mg, consistent with disulfide bond generation trends. The changes of the secondary structure of myofibrillar protein were analyzed by Infrared spectroscopy (FTIR), indicated that during the freeze-thaw cycles the spatial conformation of myofibrin made a change. Three thawing ways were treatments after 9 freeze-thaw cycles, the spectral band shifted to different wavelength regularly, and the amide A band and the amide Ⅲ band moved slightly toward the high wave number direction, and the amide Ⅰ band and the amide Ⅱ band moved to the low wave number direction. The secondary structure of the protein changed, as the sum of the contents of both α-helix and β-sheet reduced, and the sum of the contents of β-turn and random curl increased. Repeated freezing and thawing is actually a process of slow oxidation of proteins.