To design a Checklist for quality control in intensive care unit and observe the effect of clinical application. By consulting guidelines and literature, such as Critical care medicine professional medical quality control index (2015 edition), the quality control Checklist of intensive care unit was designed. It included four parts: quality control data collection, medical record quality verification, special diagnosis and treatment, and hospital infection prevention and control supervision. Every month, a doctor with a senior professional title served as the quality control director, and was responsible for the quality control of the department's medical care, including collecting data of the past 24 hours during the morning handover, discussing and registering special diagnosis and treatment behaviors that would be performed on the day, and coordinating with the nursing team leader, controlling the quality of the whole department throughout the day, such as supervising each medical staff if they had unreasonable behaviors, checking the running and discharge medical records, and inspecting the status of the staff on duty. The data in 2018, 2019 (Checklist implemented) and 2017 (Checklist not implemented) were retrospectively analyzed, including the status of admitted patients, department management information, length of intensive care unit (ICU) stay, and the incidence of three-tube infection [ventilator-associated pneumonia (VAP), catheter-related bloodstream infection (CRBSI), catheter-associated urinary tract infection (CAUTI)], and standardized mortality, etc. From 2017 to 2019, the number of patients admitted was 373, 446, and 480, with annual growth of 19.57% and 7.62% in 2018 and 2019, respectively, and an increase of 28.69% in 2019 compared with 2017. There was no statistically significant difference in the average age and acute physiology and chronic health evaluation II (APACHE II) of patients in the three years. Compared with 2017, the length of ICU stay of patients in 2018 and 2019 were significantly shortened (days: 8.99±6.12, 9.14±7.02 vs. 10.20±7.21), and the incidence of VAP, CRBSI and CAUTI were significantly reduced [VAP (cases/1 000 ventilation days): 12.97±3.60, 9.62±3.14 vs. 17.48±4.89, CRBSI (cases/1 000 catheter days): 3.75±2.19, 3.87±1.87 vs. 6.19±3.13, CAUTI (cases/1 000 catheter days): 3.29±2.18, 3.28±1.87 vs. 5.61±3.18]. The standardized mortality were also significantly reduced [(77.27±7.24)%, (70.61±7.49)% vs. (84.41±9.05)%], the number of non-compliance with hospital infection prevention per month decreased significantly (person times: 54.00±6.30, 41.08±10.76 vs. 72.08±19.68), and the number of special diagnosis and treatment per month increased significantly (person times: 1 056.67±235.27, 1 361.75±278.48 vs. 722.25±145.96), the rate of etiology submission before antimicrobial treatment [(93.21±3.68)%, (96.59±2.49)% vs. (87.86±5.28)%] and deep vein thrombosis (DVT) prevention rate [(91.13±6.36)%, (96.23±2.99)% vs. (85.58±7.68)%] were significantly improved, and all the differences were statistically significant (all P < 0.05). All medical records in the three years were Grade A, but the average scores in 2018 and 2019 were higher than those in 2017 (96.82±2.84, 96.73±2.94 vs. 93.70±3.33, both P < 0.01). Compared with 2018, the incidence of VAP, the rate of etiology submission before antimicrobial treatment, the DVT prevention rate, and the standardized mortality rate in 2019 were further improved, and the number of non-compliance with hospital infection prevention per month decreased and the number of special diagnosis and treatment per month increased, and the differences were statistically significant (all P < 0.05). The application of quality control Checklist in intensive care unit can build an effective quality control system, reduce the incidence of three-tube infection, standardized mortality and length of ICU stay, improve the quality control awareness and execution of medical staff, and promote the improvement of medical quality.