The invention relates to a new use of tRF-Leu-CAG. NSCLC is one of cancers having the highest fatality rate, and an effective early diagnosis method has important influence on treatment and prognosis of diseases. The tRF-Leu-CAG is proved to be obtained by breaking at an anticodon loop of tRNA-Leu-CAG-1 or tRNA-Leu-CAG-2 and has the length of 34 nt. The tRF-Leu-CAG is verified to have the expression level obviously up-regulated in an NSCLC cell line through a qRT-PCR method. Cell proliferation and changes of the cell cycle are detected by CCK8 and flow cell cycle analysis means, the tRF-Leu-CAG inhibitor is found out to be able to inhibit H1299 cell proliferation and also inhibit G1/S conversion to block cells in a G1 stage. A fact that the tRF-Leu-CAG inhibitor disclosed by the invention is used for preparation of NSCLC treatment drugs is reported for the first time, and certain application value is provided for preparation of the NSCLC treatment drugs in clinic.