Stick-slip is a ubiquitous motion in the hydrogen bonding network, which confers the corresponding materials with excellent toughness and strength. The experimental study of the stick-slip mechanism remains challenging because of the complexity of stress accumulation and release. An ideal system for study of this motion should comprise a defined molecular structure and chain arrangement and strong intermolecular interactions. In this study, we detected the stick-slip motion at the single-molecule level in the hydrogen bonding network of polyamide (PA) single crystals through atomic force microscopy (AFM)-based single-molecule force spectroscopy. Our results show that a stiffer force-loading device can enhance the stick capacity by increasing the fracture force and facilitating stress release. We confirm that the chain rotates while slipping and the slip distance is dependent on the unit structure of the hydrogen bonding network.