Stimuli-responsive materials have been used in biomedical applications. Composite films fabricated using polyion complexes comprising anionic and cationic polysaccharides exhibited loading and release abilities for water-soluble molecules, the release ability of which depended on the solution pH. However, the interactions between polysaccharides and loaded molecules in the film have not been evaluated. In this study, polysaccharide composite films loaded with fluorescein (FL) as a probe molecule were fabricated and the film properties, FL ionization, and release behaviour of FL were investigated. FL loading did not significantly affect the mechanical and morphological properties of the films. The release behaviour of FL was determined by the pH of the solution as well as the electrostatic interaction between polysaccharides and FL ionic structures in FL-loaded films. Furthermore, the ionic structure change of FL that remained in the film was suppressed due to interactions with polysaccharides, such as through hydrogen bonding. Additionally, the pH responsiveness of FL in the film in the dried state was evaluated. The result shows that polysaccharide composite films were swollen because of air moisture and that the diffusion of molecules inside the film accelerated. These findings are useful to understand the properties of the loaded molecules such as ionic state and diffusiveness in the films made of polyion complexes.