Objective To explore the effect of bundle intervention measures on the prevention of healthcare-associated infection(HAI) in patients with hematological malignancies(HM), and provide basis for formulating HAI preventive measures for patients with hematological malignancies. Methods By using the combination method of retrospective and prospective investigation, patients with HM in the hematology department of a hospital were selected as the research objects, those who received routine intervention from January 1, 2017 to January 31, 2018 were as control group, and those who received bundle intervention from February 1, 2018 to September 20, 2018 were as trial group, changes in incidence and site of HAI between two groups of patients before and after bundle intervention during therapeutic process were compared. Results Incidence of HAI in trial group was lower than control group (8.18%[28/342] vs 17.40%[75/431], P<0.05). Classified analysis results showed that compared with control group, incidence of HAI in patients with lymphoma in trial group decreased most obviously (28.85% vs 9.80%), followed by patients with acute leukemia (24.42% vs 13.27%). Difference in the incidence of HAI between patients with these two kinds of diseases before and after bundle intervention were both significant (both P<0.05); constituent ratios of infection sites in patients with lymphoma and acute leukemia changed significantly, constituent ratios of infection in respiratory system, oral cavity, and soft tissue decreased, while that in bloodstream increased. Conclusion The bundle intervention can reduce the incidence of HAI in patients with HM.