Disclosed is a method of manufacturing a three-dimensional semiconductor memory device including a ferroelectric thin film. The method includes forming a mold structure including interlayer dielectric layers and sacrificial layers alternately stacked on a substrate, forming channel holes penetrating the mold structure, forming vertical channel structures inside the channel holes, forming an isolation trench penetrating the mold structure and having a line shape extending in one direction, selectively removing the sacrificial layers exposed by the isolation trench, forming gate electrodes filling a space from which the sacrificial layers are removed, and performing a heat treatment process and a cooling process for the vertical channel structures.