A method for the determination of scopoletin in Tengluoning capsule by three-dimensional fluorescence combined with unfolded partial least squares coupled to residual bilinearization(U-PLS/RBL) was developed.Experiments showed that scopoletin has a strong fluorescent signal.The optimum pH of the system was 9.0,the maximum excitation wavelength of scopoletin was 375 nm,and the maximum emission wavelength was 460 nm.Excitation-emission matrices(EEM) of Tengluoning capsule samples were obtained,recording emission between 400 nm and 550 nm,exciting between 310 nm and 420 nm,for the analysis of scopoletin.An effective second-order calibration method,U-PLS/RBL(unfolded partial least-squares coupled to residual bilinearization) was used to analyze the three-dimensional fluorescent data array,and the content of scopoletin was successfully determined.The predicted U-PLS/RBL concentrations were satisfactory compared with those obtained by high performance liquid chromatography with fluorescence detection.The average recovery by U-PLS/RBL was 100.4%，which indicated that the proposed method was reliable.Compared to chromatographic method,the fluorescent method has the advantages of low operating cost and pro-environment.