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Acute myeloid leukemia (AML) blasts often have high CD135 (FLT3 receptor) expression, but its clinical impact is unclear. We analyzed CD135 expression, and the clinical characteristics and outcomes of 214 patients with de novo AML diagnosed between October 2022 and May 2024. Subsequently, we collected data on additional 78 patients, diagnosed with AML at four medical centers from June to December 2024, for external validation. The high-CD135-expression group had significantly lower CD34 surface expression (p = 0.003) and higher CD33 expression (p = 0.014) on AML blasts. The high-CD135-expression group also showed a higher frequency of NPM1 (p < 0.001) and DNMT3A (p = 0.032) mutations, but was not significantly associated with CD135 expression (p = 0.229). The patients in the high-CD135-expression group had lower initial induction therapy response rates than those in the low-CD135-expression group (p < 0.001). High CD135 expression was independently associated with poorer OS and PFS. In the subgroup of patients with high CD135 expression and FLT3-ITD mutations, those who received TKI combined with chemotherapy had significantly better OS (p = 0.007). Then we developed a prognostic nomogram incorporating CD135 expression. This model performed well both in the development cohort (area under the curve [AUC] = 0.817) and multicenter validation cohort (AUC = 0.722). CD135 expression on AML blasts is a pivotal marker that integrates molecular pathogenesis with clinical outcomes, highlighting its dual role as a prognostic indicator and therapeutic target in precision clinical approaches for AM.