Objective To explore the effect of root cause analysis (RCA) combined with procedure optimization on temperature in NICU preterm infants going out for diagnostic tests. Methods A RCA group was established, and 67 NICU preterm infants going out for diagnostic tests from January to July 2020 were tracked using RCA to investigate the causes of hypothermia, the proximal causes were found and the root causes were analyzed, then the improvement measures were developed, including resetting, merging and optimizing the procedure of going out for diagnostic tests. In August 2020 to February 2021, totally 58 preterm infants received the improved measures during going out for diagnostic tests, and the incidence of hypothermia before and after the improvement were compared. Results After implementation of the improved measures, the temperatures of the preterm infants during going out for B-mode ultrasound imaging of part of, or full abdomen, and auditory evoked potentials test were significantly improved (P<0.01 for all), and the incidence of hypothermia decreased from 37.31% before to 10.34% (P<0.01) after the implementation. Conclusion RCA combined with procedure optimization in preterm infants going out for diagnostic tests, could effectively reduce the incidence of hypothermia during going out for diagnostic tests.