The aim of our study is to develop a novel method to determine fastly and nondestructively the content of paeoniflorin and moisture in Paeoniae Radix Alba by near-infrared diffuse reflection spectroscopy. Samples with different concentrations were analyzed by HPLC and Loss on drying method; Multivariate calibration models based on PLS algorithm were developed to correlate the spectra and the corresponding values determined by the reference method. The paeoniflorin and moisture correlation coefficient (R2)of the calibration model were 0.9473 and 0.9509； Internal cross-validation mean square error( RMSECV) were 0.533 and 0.187；The correlation coefficient(R2) of the true value and prediction value from external validation were 0.9362 and 0.9440 variance of external validation( RMSEP) were 0.092 and 0.253. The results indicated that near-infrared diffuse reflection spectroscopy determination method is rapid, non-destructive, accurate and non-pollution, which can dispose the samples without complicated pretreatment. It could be applied for the rapid determination of large quantities of numbers of Paeoniae Radix Alba samples.