Objective: To investigate the characteristic and prognostic significance of leukemia stem cells associated antigens expressions including CD34, CD38, CD123, CD96 and TIM-3 in t (8;21) AML. Methods: Bone marrow samples of 47 t (8;21) AML patients were collected at diagnosis from October 2015 to April 2018 in Peking University Peoples’ Hospital, then flow cytometry method was performed to detect the expression frequencies of CD34, CD38, CD123, CD96 and TIM-3 to analyze the relationship between leukemia stem cells associated antigens expressions and relapse. Results: Of 47 t (8;21) AML patients tested, the median percentages of CD34⁺CD38^-, CD34⁺ CD38^-CD123⁺, CD34⁺CD38^- CD96⁺ and CD34⁺ CD38^- TIM-3⁺ cells among nucleated cells were 2.37%, 0.24%, 0.27% and 0.06%, respectively. All the frequencies of CD34⁺CD38^-, CD34⁺CD38^-CD123⁺, CD34⁺CD38^-CD96⁺ and CD34⁺ CD38^-TIM-3⁺ cells had no impact on the achievement of CR after the first course of induction. All higher frequencies of CD34⁺CD38^-, CD34⁺CD38^-CD123⁺, CD34⁺CD38^-CD96⁺ cells were related to higher 2-year CIR rate. Whereas, the frequency of CD34⁺ CD38^- TIM-3⁺ cells had no impact on CIR rate. Both high frequency of CD34⁺ CD38^- cells and the high level of minimal residual diseases (patients with <3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy) were independent poor prognostic factors of CIR[P=0.025, HR=6.9 (95%CI 1.3-37.4) ; P=0.031, HR=11.1 (95%CI 1.2-99.2) ]. Conclusion Different leukemia stem cells associated antigens had distinct prognostic significance in t (8;21) AML. High frequencies of CD34⁺ CD38^-, CD34⁺ CD38^- CD123⁺ and CD34⁺CD38^-CD96⁺ cells at diagnosis predicted relapse in patients with t (8;21) AML.