Both haploidentical hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) exhibit strong graft-versus-leukemia (GVL) effect. However, the role of prophylactic DLI following haploidentical HSCT remains unclear. Here, 34 patients with high-risk acute leukemia who underwent low-dose anti-T-lymphocyte globulin-Fresenius (ATG-F)-based myeloablative haploidentical HSCT and prophylactic modified DLI (pro-DLI) were well-matched with patients without pro-DLI. The 5-year overall survival (OS) (67.8% versus 41.3%, P < 0.01) and leukemia-free survival (LFS) (64.6% versus 33.9%, P < 0.01) of pro-DLI cohort were superior to the control cohort. A slightly higher GVHD-free/relapse-free survival was found in the pro-DLI cohort (32.8% versus 16.3%, P = 0.32). The 5-year cumulative incidence of relapse of the pro-DLI recipients was significantly lower than that of the control cohort (14.7% versus 49.3%, P = 0.01). The cumulative incidence of grades II-IV and III-IV acute GVHD at 100 days after pro-DLI was 17.6% and 9.1%, respectively. There was no difference between the two cohorts in terms of the cumulative incidence of chronic GVHD and non-relapse mortality. Data from the multivariate analysis demonstrated that pro-DLI was an independent protective variable for LFS (P = 0.01, hazard ratio {HR} = 0.35), OS (P = 0.01, HR = 0.32), and relapse (P = 0.02, HR = 0.33). Taken together, we demonstrate that pro-DLI after ATG-F-based HSCT effectively decreases the risk of relapse and improves long-term survival of patients with high-risk acute leukemia without increasing treatment toxicity.