Exploiting the uses of proteins and polysaccharides is of increasing interest because the combination of the attributes of these two types of biopolymers can produce small emulsion droplets with good physical stability. The objective of this study was to evaluate the effect of adding chitosan to a bovine serum albumin (BSA) solution at different pH values, and the consequent conformational changes were monitored using UV-vis absorption spectroscopy and fluorescence spectroscopy. Spectroscopic observations were further combined with rheological analysis results to illustrate the interaction mechanism. The results showed that the pH and BSA/chitosan ratio significantly affected the interaction between BSA and chitosan. Additionally, the microenvironment and the secondary structure of BSA were changed with the addition of chitosan, and chitosan was found to quench the intrinsic fluorescence of BSA. The experimental results provide theoretical guidance for the design of new ingredients for the food industry.