Abstract: Cordyceps militaris is a traditional medicinal mushroom that helps to revitalize and increase human health. Cordycepin is the main active compound of Cordyceps militaris, which plays an active role in the inhibition of cancer cell growth and in the prevention of virus infection. However, there has been little research on the effects of the extraction process on the level of cordycepin. In this study, we investigated the effects of temperature, the concentration of solvent, and time of extraction on the level of cordycepin in the extract of Cordyceps militaris fruiting bodies that are cultured at the Institute of Applied Technology, Thu Dau Mot University. The levels of cordycepin in the extract were evaluated by ultra-fast liquid chromatography (UFLC). The results showed the highest level of cordycepin was obtained at 1.10g/kg fresh fruiting bodies using 70 ͦEtOH, at 80oC for 2h. This level was equivalent to 6.97g/kg of cordycepin per dried fruiting bodies. These findings provide the basis for subsequent research on the pharmaceutical effects of fruiting body extract of Cordyceps militaris.