•The molecular recognition of cordycepin arabinoside in Cordyceps militaris by MS2 fragmentation rule.•A quantitative analysis method of cordycepin and its arabinoside was established by HPLC-MS/MS.•The cordycepin and its arabinoside were determined in fruiting body and pupa during growth process.•Cordyceps militaris as the optimum raw material of functional foods during 60th to 70th days. Cordyceps militaris is an edible fungus that is widely used as a functional food in many countries. In order to objectively evaluate its nutritional value, free and glycosidic cordycepins need to be analyzed. The cordycepin arabinoside molecule was recognized by the MS2 fragmentation rule, and both cordycepin and its arabinoside were quantitatively analyzed in the fruiting body and pupa of Cordyceps militaris by high-performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS). The method had good linear regression (R2 = 0.9999), with a detection limit of 0.021 ng/mL. The recovery range was 94.32–103.09% in the fruiting body and pupa. The content of cordycepin and its arabinoside showed an upward trend with growth, and the total contents reached the highest level at the mature stage (60-70th day) without mildew. This study provides a useful reference for the evaluation and application of Cordyceps militaris as a functional food resource.