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The effect of freeze-thaw cycling on the quality of pre-fermented red bean steamed bun (RBSB) dough and the cooked product were explored. For frozen dough samples, the changes in water distribution, frozen water content, starch crystallinity, layered structure, and protein secondary structure of the dough during freeze-thaw cycling were analyzed. The textures, colors, and relative amino acids contents of RBSBs, which were steamed from frozen dough, were analyzed. The results showed that the transverse relaxation time of weakly bound water,T22,increased as the number of freeze–thaw cycles increased, and the freezable water content reached its highest value (14.68%) after 1 cycle. The relative crystallinity of starch was increased by 13.6% compared with the control after 4 freeze-thaw cycles. The lamellar structure of starch increased by 4.33% after more than 5 freezing cycles. Freeze-thaw treatments caused significant and irregular changes in the secondary structure of the frozen dough. Freeze-thaw cycling accelerated an increase in the hardness of RBSB products. The brightness (L*) of RBSBs decreased significantly with increases in the number of freeze-thaw cycles, and these cycles also affected the amino acid contents of RBSBs. [ABSTRACT FROM AUTHOR]