Objective To investigate the expression and significance of matrix metalloproteinases (MMPs) and toll-like receptor 9 (TLR9) in patients with acute leukemia. Methods A total of 44 patients with acute lymphoblastic leukemia (ALL) were enrolled in the ALL group, 30 patients with acute myeloid leukemia (AML) were included in AML group, and 44 healthy individuals undergoing physical examination during the same period were included in the control group. Serum levels of MMP-2, MMP-7, and MMP-9 were measured by enzyme-linked immunosorbent assay (ELISA) in all three groups. The expression of TLR9 mRNA and TLR9 protein in peripheral blood mononuclear cells (PBMCs) was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Spearman's rank correlation analysis was used to explore the correlations of the malignancy of different clinical subtypes of acute leukemia with the expression levels of MMPs and TLR9 protein. Results Serum levels of MMP-2, MMP-7, and MMP-9 were significantly higher in the ALL and AML groups compared to the control group, with the AML group showing higher levels than the ALL group (P < 0.01). The relative expressions of TLR9 mRNA and TLR9 protein in PBMCs in both the ALL and AML groups were significantly lower than those in the control group, and the AML group had lower expression than the ALL group (P < 0.01). Spearman's rank correlation analysis revealed positive correlations of serum levels of MMP-2, MMP-7, and MMP-9 with the malignancy of both ALL and AML (P < 0.05), while the relative expression of TLR9 protein was negatively correlated with the malignancy of both ALL and AML (P < 0.05). Conclusion Serum levels of MMP-2, MMP-7, and MMP-9 are significantly elevated in patients with acute leukemia, while TLR9 expression is significantly reduced in PBMCs. The abnormal overexpression of MMPs may promote extramedullary infiltration and immune escape of acute leukemia cells by inhibiting TLR9 expression, thereby accelerating the malignant progression of the tumor.