The aim of this study was to label Bovine Serum Albumin (BSA) by Rhodamine B Isothiocyanate (RBITC) and test its stability in vivo. RBITC-BSA was labelled by the improved Marshall’s method, scanned at full wavelength, and the RBITC:BSA ratio was calculated. Blood and urine samples were obtained from rabbits injected with the RBITC-BSA complex and SDS-PAGE analysis was performed. Full wavelength scanning showed that the labelled RBITC-BSA complex possessed features of both RBITC and BSA, and the RBITC:BSA ratio was 1:80. Furthermore, the RBITC-BSA complex was stable in vivo. Thus, we developed a stable RBITC-BSA complex with high specificity and sensitivity, which could be used as a tracer molecule to study protein transportation and vascular permeability.