PEGylation can modify the physicochemical properties of native chitosan and improves its water solubility. PEGylated chitosan has been widely used as a gene/drug delivery vector by forming a polyelectrolyte complex (PEC) in biomedical engineering. The molecular interactions of PEGylated chitosan play a critical role in forming the core–shell structure of the complexes. In this work, we systematically investigated the cohesive interaction between PEGylated chitosan films using a surface forces apparatus (SFA) under different solution conditions, and the corresponding morphology change was characterized using atomic force microscopy (AFM). The force measurements demonstrated that the cohesion could be enhanced by increasing the contact time and the PEGylation degree, but could be weakened by increasing the solution pH, which is closely related to the morphology change of the PEGylated chitosan films. The strong cohesion of PEGylated chitosan, as compared to that of native chitosan, is primarily attributed to improved polymer solubility and flexibility, and enhanced formation of hydrogen bonds between the polymer chains. In addition, continuously increasing the PEGylation degree was found to be less effective in further strengthening the cohesion at relatively high pH (e.g., pH ∼ 8.5), which is most likely due to the repulsion originating from the formation of dense hydration PEG shells. Our results provide useful nanomechanical insights into the fundamental understanding of the interaction mechanism of PEGylated chitosan, with implications for the development of novel and effective gene/drug carriers in bioengineering.