Three-dimensional synchronous fluorescence spectrum of prepared rehmannia root from different regions were measured and feature parameters were extracted. It can be found that the effective fluorescent compositions of prepared rehmannia root from different regions are similar. The relationships between the spectrum-effect were established. This study can provide reference for clinicaldosage. First order derivative and second order derivative of prepared rehmanniaroot from different regions can be got by programming respectively. Magnifing synchronous emission spectrum of the shoulder strap, the subtle differences of the spectrum can be distinguished and the method, derivative synchronous fluorescence spectrum, to identify different regions of prepared rehmannia root are acquired.