Objective To explore the impact of multi-department collaboration mode on improving the positive rate of blood culture and reducing the intensity of antimicrobial use. Methods Patients who underwent blood culture in the key internal medicine departments for infection management from January to December 2020 were selected as the research subjects. The blood culture and antimicrobial use were analyzed. Patients from January to June 2020 were the control group, while patients from July to December 2020 were the intervention group. Intervention measures for blood delivering were implemented starting in July 2020. Ten quality control indicators related to blood culture, such as the average delivering time of blood culture specimens, were set up to assess the key departments monthly, and the improvement of the 10 indicators were observed. Blood specimen delivering rate before antimicrobial use, blood culture delivering modes, specimen contamination rate and positive rate, as well as antimicrobial use rate of hospitalized patients before and after the intervention through multi-department collaboration management mode were compared. Results A total of 16 246 blood culture specimens were delivered for detection, including 7 512 in the control group and 8 734 in the intervention group. The delivering rate of two or more sets of blood delivering in the intervention group (24.91%) was higher than that in the control group (15.04%), and the difference was statistically significant (P < 0.001). The average delivering time of the intervention group was 18.56 minutes (reaching the quality control goal), the average machine operation time was 18.83 minutes (reaching the quality control goal), and the average reporting time of positive results to clinical department was 148.28 minutes (not reaching the quality control goal, ≤1 hour). The average delivering time, machine operation time, and reporting time for positive results to clinical departments in the intervention group were all higher than those in the control group, and the differences were all statistically significant (all P < 0.001). The accuracy rate of collection opportunity of blood culture (98.33% vs 74.67%), accuracy rate of blood collection method (82.67% vs 67.67%), and accuracy rate of correct treatment for clinical department with positive blood culture (70.67% vs 62.00%) in the intervention group were all higher than those in the control group, and the differences were statistically significant (all P < 0.05). The positive rate of blood culture in the intervention group was higher than that in the control group (9.43% vs 6.56%); the contamination rate was lower than that in the control group (0.77% vs 1.28%); the unqualified rate was lower than that of the control group (1.19% vs 2.18%), all the differences were statistically significant (all P < 0.05). After the intervention, the antimicrobial use intensity in the intensive care medicine department, hematology department, infectious disease department, and the neurology department decreased by 2.78%, 42.36%, 27.82%, and 39.47%, respectively, while the pulmonary and critical care medicine department increased by 8.10%. Conclusion The multi-department collaboration mode has a good impact on improving the positive rate of blood culture and reducing the intensity of antimicrobial use in the internal medicine departments.