Understanding the folding pattern of a single polymer chain within its single crystal will shed light on the mechanism of crystallization. Here, we use the combined techniques of atomic force microscopy (AFM)-based molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations to study the folding pattern of a polyethylene oxide (PEO) chain in its single crystal. Our results show that the folding pattern of a PEO chain in the crystal formed in dilute solution follows the adjacent re-entry folding model. While in the crystal obtained from the melt, the nonadjacent folding with large and irregular loops contributes to big force fluctuations in the force extension curves. The method established here can offer a novel strategy to directly unravel the chain-folding pattern of polymer single crystals at single-molecule level.