In order to determine the content of trehalose in mushroom, a procedure of trehalose extraction from Cordyceps militaris was developed. The results proved that the optimal method for extraction of trehalose was as follows: 1g of powder of Cordyceps militaris fruiting bodies was extracted by reflux in 100mL 90% ethanol for 1h. Then a high performance liquid chromatography (HPLC) was developed for the determination of trehalose. By use of SUGAR SP0810 column (300mm′8mm) as the chromatographic column and purified water as mobile phase, trehalose was separated completely at the refractive index detector with the flow rate of 0.5mL/min at the temperature of 70℃. The injection volume was 10μL. The methodological study showed that the method was sensitive, accurate, reproducible and successfully applied to analyze the trehalose in Cordyceps militaris.