The present work describes a simple, sensitive, selective, and eco-friendly spectrofluorimetric method for bismuth determination in wastewaters and dyed scalp hair. The method is based upon fluorescence enhancement accompanying a direct reaction between rhodamine B (RB) and bismuth(III) ions, and the formed complex was then extracted from aqueous medium into a chloroform phase using dispersive liquid–liquid microextraction (DLLME). The proposed method was capable of bismuth determination even in the presence of 20 common ions in 100-fold excess over the bismuth. The plot of fluorescence enhancement measured at λ_ex/em = 552/572 nm in the organic phase versus bismuth concentration in an aqueous solution was linear over the range of 0.5–100 μg L⁻¹. The LOD and LOQ were found to be 0.16 and 0.532 μg L⁻¹, respectively. The accuracy of the method was verified by analyzing bismuth in a certified standard reference material (SRM-3106). The method was also successfully applied for the analysis of bismuth in wastewaters, and dyed scalp hair samples, and the results were successfully compared with the inductively coupled plasma-mass spectrometry (ICP-MS) results. Furthermore, the structure of the newly synthesized complex was confirmed with the aid of spectroscopic measurements (UV-Vis, IR, fluorescence), and elemental and thermal analyses. These measurements indicated that the enhanced fluorescence response was attributable to coordination of Bi³⁺ ions with the RB reagent.