This study examined the effect of herring antifreeze protein (hAFP) combined with chitosan magnetic nanoparticles (CS@Fe3O4), applied through vacuum impregnation, on protein conformation and moisture state of red sea bream during freeze-thaw cycles. DSC and dynamic rheology were used to analyze physicochemical changes. Raman, intrinsic fluorescence and second derivative UV spectrometry were used to explore the structural changes of myofibrillar proteins. Surface hydrophobicity, particle size and zeta-potential were used to analyze protein aggregation and denaturation. AV, POV, and TBA were used to determine the degree of lipid oxidation. LF-NMR and MRI were used to test water migration and distribution. The results indicated that pretreatment of red sea bream through vacuum impregnation with hAFP combined with CS@Fe3O4 solutions could minimize freeze-thaw damage, prevent protein and lipid oxidation. Therefore, these results confirm the importance of hAFP as a valuable food additive in frozen foods.