Gelatin is a highly versatile hydrocolloid that plays a significant role in various applications as both a gelling and emulsifying agent in the food and pharmaceutical sectors. In the food industry, the mechanisms by which gelatin stabilizes emulsions have been extensively studied. Nonetheless, the dynamic changes, specifically the reconfiguration of the gelatin layer at the interface when pH changes occur in situ, such as during gastric transit, are frequently overlooked. In this study, we aimed to establish the relationship between dynamic pH changes and the structural reconfiguration of adsorbed gelatin on a model colloidal particle using microfluidics and optical tweezers. Microfluidics allows for variation in the solution environment, while optical tweezers enable the measurement of the hydrodynamic layer thickness of adsorbed gelatin in the presence of a flow field. When a 50 ppm gelatin solution prepared at pH 8.5 (isoelectric point) was injected, a temporal increase in the hydrodynamic layer thickness was observed (Fig. 1), indicating the adsorption of gelatin. Moreover, when a low pH solution was injected, an increase in the hydrodynamic layer thickness was demonstrated. This suggests a pronounced swelling of the gelatin layer at the interface, which is attributed to an increase in the net positive charge density, enhancing electrostatic repulsion between the adsorbed polymer chains. When the solution pH was changed back to high pH, a decrease back to the original adsorbed layer thickness was observed. Thus, this study provides important insights into the structural reconfiguration of the adsorbed gelatin onto a single colloidal interface.