Here we report the use of a Langmuir isotherm model to analyze and better understand the dynamic adsorption and desorption behavior of single fluorophore molecules at the surface of a hydrogen nanobubble supported on an indium tin oxide (ITO) electrode. Three rhodamine dyes, rhodamine 110 (R110, positively charged), rhodamine 6G (R6G, positively charged), and sulforhodamine G (SRG, negatively charged) were chosen for this study. The use of the Langmuir isotherm model allows us to determine the equilibrium constant and the rate constants for the adsorption and desorption processes. Of the three fluorophores used in this study, SRG was found to have the greatest equilibrium constant. No significant potential dependence was observed on the adsorption characteristics, which suggests the nanobubble size, geometry, and surface properties are relatively constant within the range of potentials used in this study. Our results suggest that the use of the Langmuir isotherm model is a valid and useful means for probing and better understanding the unique adsorption behavior of fluorophores at surface-supported nanobubbles.