The invention discloses a novel molecular marker and its application in preparation of a kit for the diagnosis and prognosis of head and neck cancer. The RNA sequences of the novel molecular marker are shown in SEQ ID NO:1-SEQ ID NO:48. Based on the marker of the invention, a mathematical model for the diagnosis of head and neck cancer is constructed; the model has high sensitivity, good specificity, AUC up to 0.981, and good diagnostic effect. In addition, these 48 tRF fragments can also be used as molecular markers for head and neck cancer classification and prediction of patient survival; in the test data, according to the expression of tRFs, head and neck cancer tumor samples were clustered into 4 subtypes, and analyzed by survival curves It shows that there are significant differences in the survival periods of these subtypes; the novel tRF molecular marker disclosed in the present invention has the characteristics of good diagnostic indicators, can be effectively used in the diagnosis, classification and prognosis of head and neck cancer, and has high clinical application and promotion value.