OBJECTIVE： To determine the content of paeonol in Cynanchum paniculatum by three-dimensional fluorescence coupled with ATLD algorithm. METHODS： The trilinear model was established. The principle of least square was used for ATLD.  The component value was fitted by core consistency diagnosis， and inner filter effect was corrected by mathematical correction method. Fluorescence scanning condition included excitation wavelength of 250-400 nm， emission wavelength of 410-600 nm， wavelength interval of 5 nm， slit width of 5.0/5.0 nm， scanning speed of 1 200 nm/min. Determination and absorption spectrum condition included scanning wavelength of 250-600 nm， scanning speed of 600 nm/min； Al（Ⅲ） was used as the sensitizer to increase the fluorescence intensity of paeonol. The content of paeonol was determined by HPLC. RESULTS： The linear range of paeonol was 0.132-1.188 μg/mL（r＝0.999 9）. RSDs of precision， stability and reproducibility tests were less than 3.0％. The recoveries were 100.1％-104.7％（RSD＝2.39％，n＝6）. The analysis spectrum of paeonol was almost completely overlapped with the actual spectrum.  The results of HPLC method are very similar to those of ATLD algorithm. CONCLUSIONS： The three-dimensional fluorescence coupled with ATLD algorithm is simple， rapid， efficient and accurate， and can be used for the qualitative and quantitative analysis of complex system.