The invention belongs to the field of fingerprints of Chinese herbal medicines and particularly relates to a construction method of a protein fingerprint of fresh cordyceps sinensis and an application of the method in identification of the Chinese herbal medicine, namely, cordyceps sinensis. According to the method, a sample is pretreated and then determined with an LC-Q-TOF-MS technique under the following chromatographic conditions: a chromatographic column is Welch Xtimate C4; a filling agent is tetralkyl silane bonded silica gel; a mobile phase comprises components A and B, the component A is 0.1% formic acid-water and the component B is 0.1% formic acid-acetonitrile; gradient elution is performed. A result indicates that compared with the prior art, the construction method has the advantages as follows: the protein fingerprint of cordyceps sinensis is constructed with the LC-1-TOF-MS technique initially, the optimal parameters of a sample protein extraction condition and an analysis condition are determined, the sensitivity is high, the separation effect is good, and the accuracy and the reliability of the result are improved. Finally, the protein fingerprint is used for true and false identification of the fresh cordyceps sinensis and can provide a reference for quality control of cordyceps sinensis.