Background: Cultured Cordyceps militaris is very popular. Objective: To gain dynamic insight into activity markers in fruiting body of Cordyceps militaris (C. militaris) in Bombyxmori (B. mori), also named silkworm. Methods: The development stages of samples at 3, 9, 12, 19, 27, and 33 days after inoculation (DAI) were collected. HPLC coupled with diode array detection and evaporative light-scattering detection method (HPLC-DAD-ELSD) was used to determine eight makers, including six nucleosides and two carbohydrates from the samples. Results: C. militaris cultured 33 DAI with fifth star silkworm larva could accumulate higher levels of cordycepin (13.43 mg/g) than the highest reported cordycepin (8.57 g/L). The contents of cordycepin, adenosine, and trehalose were gradually increased with the formation of C. militaris fruiting bodies on silkworm larva, while mannitol was decreased. The change of guanosine was similar to uracil. Conclusions: Results suggested that mannitol could be accumulated in a short period during mycelium growth and could metabolize and transform into energy store and trehalose during fruit body formation. The inosine in the insect was completely utilized and transformed. The synergistic formation of cordycepin and adenosine or differences in metabolized pathways are a great possibility according to the same trend. Highlights: This research offered some reference to further find a certain regularity or metabolic mechanism.