Abstract: Object: To explore the optimal extraction conditions of cordycepin in multigrain cordyceps militaris hyphae symbionts,the effect of varied lighting duration on cordycepin was compared,the purpose was to provide a basis for the optimization of its technological conditions and comprehensive utilization.Methods: The conditions of waterbath extraction of cordycepin were determined by single factor experiments combined with response surface methodology,the content of cordycepin was determined by high performance liquid chromatography,SPSS 17.0software was applied for statistical analysis. Results: The results showed that the optimal extraction conditions were: extraction time 6.59 h,extraction temperature 81.9 ℃,solid- liquid ratio 1∶90.The contents of cordycepin were6.0143 mg / g under this condition. The contents of cordycepin were 3.8988 ~ 11.9629 mg / g in different lighting duration and were significantly different( p = 0.000).Conclusions: The optimal conditions of waterbath extraction of cordycepin were determined and the contents of cordycepin were significantly different under varied lighting duration,which can provide the reference basis for further optimization of culture conditions of multigrain cordyceps militaris hyphae symbionts.