Chitosan is a cationic biopolymer that has many potential applications in the food industry because of its unique nutritional and physiochemical properties. Many of these properties depend on its ability to interact with anionic surface-active molecules, such as phospholipids, surfactants, and bile acids. The purpose of this study was to characterize the interaction between chitosan and a model anionic surfactant (sodium dodecyl sulfate, SDS) using isothermal titration calorimetry (ITC), surfactant-selective electrode (SSE), and turbidity measurements. ITC and SSE indicated that SDS bound strongly to chitosan via a highly exothermic interaction. The turbidity measurements indicated that chitosan formed insoluble complexes with SDS that strongly scattered light. The chitosan bound approximately 4 mM of SDS per 0.1 wt % chitosan before becoming saturated with surfactant. The SDS−chitosan interaction was weakened appreciably by the presence of 100 mM NaCl, which suggested that it was electrostatic in origin. This study provides information about the origin and characteristics of molecular interactions between chitosan and anionic surface-active lipids that may be useful for the rational design of chitosan-based food ingredients with specific nutritional and functional characteristics, e.g., cholesterol lowering or fat replacement.