Abstract: The purpose of this study was to determine the quantitative relationship between the intestinal absorption rate and concentration of rhodamine B, and to evaluate the feasibility of indocyanine green as a reference for intestinal perfusion in vivo. The absorption behavior of rhodamine B at different concentrations was systematically explored through in vivo fluorescence imaging and in vivo one-way intestinal perfusion experiments after gavage in rats, combined with fluorescence intensity measurement and pharmacokinetic data analysis. At the same time, indocyanine green was used as a volume marker to verify the feasibility and reliability of it instead of traditional phenol red for volume correction in intestinal perfusion experiments. Studies have found that the intestinal absorption of rhodamine B is influenced by passive diffusion and carrier-mediated transport. The Michaelis constant is about 3.5 mg/L, and the transporter saturation effect is significant at high concentrations. When the concentration of indocyanine green is between 0.5 and 4.0 mg/L, there is a linear relationship between the concentration and the fluorescence intensity. The recovery rate in the intestinal perfusion experiment reaches 98.5±3.1%, and it can effectively avoid the interference of the traditional markers phenol red and rhodamine B in traditional ultraviolet detection methods, providing a new solution to improve the accuracy of the intestinal perfusion experiment. These results not only fill the gap in the research on the absorption kinetics of rhodamine B in vivo, but also provide important experimental basis and theoretical support for the development of in vivo intestinal perfusion technology.