Aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a non-enzymatic component required for the multi-tRNA synthetase complex. While exon 2 skipping alternatively spliced variant of AIMP2 (AIMP2-DX2) compromises AIMP2 activity and is associated with carcinogenesis, its clinical potential awaits further validation. Here, we found that AIMP2-DX2/AIMP2 expression ratio is strongly correlated with major cancer signaling pathways and poor prognosis, particularly in acute myeloid leukemia (AML). Analysis of a clinical patient cohort revealed that AIMP2-DX2 positive AML patients show decreased overall survival and progression-free survival. We also developed targeted RNA-sequencing and RNA-smFISH tools to quantitatively analyze AIMP2-DX2/AIMP2 ratios at the single-cell level. By subclassifying hematologic cancer cells based on their AIMP2-DX2/AIMP2 ratios, we found that downregulating AIMP2-DX2 sensitizes cells to anti-cancer drugs only for a subgroup of cells while it has adverse effects on others. Collectively, our study establishes AIMP2-DX2 as a potential biomarker and a therapeutic target for hematologic cancer.