The partial tandem duplication of histone-lysine N-methyltransferase 2A (KMT2A-PTD) is an important genetic alteration in acute myeloid leukemia (AML) and is associated with poor clinical outcome. Accurate and rapid detection of KMT2A-PTD is important for outcome prediction and clinical management, but next-generation sequencing-based quantitative research is still lacking. In this study, we developed a targeted RNA-based next-generation sequencing panel, together with single primer enrichment and unique molecular identifiers, to identify KMT2A-PTD as well as AML-related gene fusions and other driver mutations. Our panel showed high sensitivity, accuracy, and reproducibility in detecting the fusion ratio of KMT2A-PTD. The mutation profile of KMT2A-PTD-positive patients with AML was characterized and different distribution patterns of driver mutations were found according to KMT2A-PTD fusion ratio level. Survival analyses revealed that the fusion ratio of KMT2A-PTD did not affect clinical outcome, but a novel molecular combination, namely, KMT2A-PTD/DNMT3A/FMS-like tyrosine kinase 3-internal tandem duplication, was associated with poor prognosis. Finally, it was shown that the dynamic changes in the KMT2A-PTD fusion ratio were consistent with the overall process of disease progression. In summary, we applied the unique molecular identifier-based RNA panel to quantitatively detect KMT2A-PTD and elucidate its clinical relevance, which complemented the integrative network of various genetic alterations in AML.