Despite being the leading cause of lung cancer-related deaths, the underlying molecular mechanisms driving metastasis progression are still not fully understood. Transfer RNA-derived fragments (tRFs) have been implicated in various biological processes in cancer. However, the role of tRFs in lung adenocarcinoma (LUAD) remains unclear. Our study identified a tRF, tRF-Val-CAC-024, associated with the high-risk component of LUAD, through validation using 3 cohorts. Our findings demonstrated that tRF-Val-CAC-024 acts as an oncogene in LUAD. Mechanistically, tRF-Val-CAC-024 was revealed to bind to aldolase A (ALDOA) dependent on Q125/E224 and promote the oligomerization of ALDOA, resulting in increased enzyme activity and enhanced aerobic glycolysis in LUAD cells. Additionally, we provide preliminary evidence of its potential clinical value by investigating the therapeutic effects of tRF-Val-CAC-024 antagomir-loaded lipid nanoparticles (LNPs) in cell-line-derived xenograft models. These results could enhance our understanding of the regulatory mechanisms of tRFs in LUAD and provide a potential therapeutic target.