This study reports on the optimization of the extraction conditions of cordycepin from Cordyceps militaris by using ultrasonication. For this purpose, the orthogonal experimental design was used to investigate the effects of factors on the ultrasonic-assisted extraction (UAE). Four factors: extraction time (min), ethanol concentration (%), extraction temperature (°C) and extraction frequency (kHz), were studied. The results showed that the highest cordycepin yield of 7.04 mg/g (86.98% ± 0.23%) was obtained with an extraction time of 60 min, ethanol concentration of 50%, extraction temperature of 65 °C and extraction frequency of 56 kHz. It was found that the cordycepin extraction yield increased with the effect of ultrasonication during the extraction process. Therefore, UAE can be used as an alternative to conventional immersion extraction with respect to the recovery of cordycepin from C. militaris, with the advantages of shorter extraction time and reduced solvent consumption.