BackgroundTransfer (t)RNA-derived small RNA (tsRNA), generated from precursor or mature tRNA, is a new type of small non-coding RNA (sncRNA) that has recently been shown to play a vital role in human cancers. However, its role in laryngeal squamous cell carcinoma (LSCC) remains unclear.MethodsWe elucidated the expression profiles of tsRNAs in four paired LSCC and non-neoplastic tissues by sequencing and verified the sequencing data by quantitative real-time PCR (qRT–PCR) of 60 paired samples. The tyrosine-tRNA derivative tRFTyrwas identified as a novel oncogene in LSCC for further study. Loss-of-function experiments were performed to evaluate the roles of tRFTyrin tumorigenesis of LSCC. Mechanistic experiments including RNA pull-down, parallel reaction monitoring (PRM) and RNA immunoprecipitation (RIP) were employed to uncover the regulatory mechanism of tRFTyrin LSCC.ResultstRFTyrwas significantly upregulated in LSCC samples. Functional assays showed that knockdown of tRFTyrsignificantly suppressed the progression of LSCC. A series of mechanistic studies revealed that tRFTyrcould enhance the phosphorylated level of lactate dehydrogenase A (LDHA) by interacting with it. The activity of LDHA was also activated, which induced lactate accumulation in LSCC cells.ConclusionsOur data delineated the landscape of tsRNAs in LSCC and identified the oncogenic role of tRFTyrin LSCC. tRFTyrcould promote lactate accumulation and tumour progression in LSCC by binding to LDHA. These findings may aid in the development of new diagnostic biomarkers and provide new insights into therapeutic strategies for LSCC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s11658-023-00463-8.