Compared to other immune checkpoint molecules, T cell immunoglobulin domain and mucin domain-3 (TIM-3) is highly expressed on natural killer (NK) cells, but its functional role and prognostic significance in acute myeloid leukemia (AML) remains unclear. This study aims to evaluate the role of TIM-3 expression on the cytotoxic and killing capacity of NK cells and its prognostic significance in AML. AML public single-cell RNA sequencing (scRNAseq) data were used to analyze the correlation of transcript levels between (encoding TIM-3) and cytotoxic molecules in NK cells. NK cells from the bone marrows of seven newly diagnosed AML patients and five healthy donors (HDs) were stimulated in vitro and cell-killing activity was evaluated. A total of one hundred and five newly diagnosed adult AML patients and seven HDs were tested the expression of TIM-3 and cytotoxic molecules on the bone marrow NK cells by multi-parameter flow cytometry (MFC). Both scRNAseq and MFC analysis demonstrated that TIM-3 expression on NK cells was positively related to the levels of perforin (PFP) and granzyme B (GZMB) (all < 0.05) in AML. It was PFP and GZMB but not the TIM-3 level that was related to NK-cell-killing activity against K562 cells ( = 0.027, 0.042 and 0.55). A high frequency of TIM-3 NK cells predicted poorer relapse-free survival (RFS) and event-free survival (EFS) ( = 0.013 and 0.0074), but was not an independent prognostic factor, whereas low GZMB levels in TIM-3 NK cells independently predicted poorer RFS ( = 0.0032). TIM-3 expression on NK cells is positively related to PFP and GZMB levels but has no relation to cell-killing activity in AML, and low GZMB levels in TIM-3 NK cells in the diagnostic bone marrows predicts poor outcomes. This study lays a theoretical foundation for the clinical application of immune checkpoint inhibitor treatment.