Investigation of the folding pattern of a single polymer chain in its crystal is of significant importance toward the understanding of the fundamental crystallization and melting as well as in engineering new materials.Here,we use the combined techniques of atomic force microscopy(AFM)-based single-molecule force spectroscopy(SMFS)and steered molecular dynamics(SMD)simulations to pull a single polyethylene oxide(PEO)chain out of its single crystal,respectively.From these data,the folding pattern of polymer chains in its spiral crystal can be reconstructed.Our results show that folding pattern of a PEO chain in the crystal obtained from dilute solution is highly regular and loops on the crystal surface is relatively small,supporting the adjacent re-entry folding model.While in the crystal formed in the melt,the non-adjacent folding with large and irregular loops contributes to big force fluctuations in the force-extension curves.These results indicate that the combined techniques represent novel experimental tools to directly unravel the chain folding pattern of polymer single crystals at single-molecule level.