Abstract: Objective:To develop a matrix solid-phase dispersion (MSPD) -high performance liquid chromatography ( HPLC) for simultaneous determination of uracil, adenine, thymine, uridine, adenosine, thymidine, cytidine, guanosine and cordycepin in natural Cordyceps sinensis, cultured Cordyceps militarris mycelia and Cordyceps militarris fruiting bodies. Methods:Nine nucleosides and nucleobases were extracted by MSPD and determined by HPLC. Results:The experimental conditions for the MSPD were optimized. Florisil was used as dispersant, petroleum ether as washing solvent and methanol as elution solvent. The ratio of Florisil to sample was selected to be 4∶1. The calibration curve had good linear relationship (r>0.9997) . The limits of detection and quantification were in the range of 12.4⁷9.4ng·mL-1and 41.2²64.6ng·mL-1, respectively. Intra- and interday precision were lower than 7.28%. The recoveries were between 81.54% and 95.77%. Conclusion:The present method was simple, and consumed less sample. The present MSPD-HPLC method should be applied for simultaneous extraction of nucleosides and nucleobases in herbal materials, pharmaceutical products and biological drugs.