Objective To evaluate the effect of evaluation and feedback after comprehensive monitoring on the operation and maintenance of laminar airflow operating rooms. Methods Technical code for construction of hospital clean operating department was used as the monitoring and judgment standard, from February to May 2017, 81 laminar airflow operating rooms in 34 hospitals in Yunnan Province were conducted baseline comprehensive monitoring, monitoring results and rectification suggestions after evaluation were fed back to each hospital, comprehensive monitoring was carried out again from November 2017 to March 2018, the baseline monitoring results were compared with the monitoring results after evaluation and feedback. Results Compared with the baseline level, the monitoring results of dust particle with particle diameter ≥0.5 μm and ≥5.0 μm as well as planktonic bacteria in laminar airflow operating rooms in secondary and tertiary hospitals were not significantly different (both P>0.05). After evaluation and feedback, the qualified rates of monitoring on cross-section wind speed, non-uniformity of wind speed β and blind zone of wind speed were all higher than those of baseline monitoring results, differences were all statistically significant (all P < 0.05); the qualified rates of temperature, relative humidity, air change times, static pressure difference, noise and illumination before and after evaluation and feedback were all not significantly different (all P>0.05). The proportion of obstacle to rectification of "unprofessional maintenance personnel" in laminar airflow operating rooms in secondary hospitals was higher than that in tertiary hospitals, difference was significant (P=0.014). Conclusion The evaluation and feedback after comprehensive monitoring on laminar airflow operating rooms can improve the effect of operation and maintenance of laminar airflow operating rooms, comprehensive monitoring on laminar airflow operating rooms should be carried out regularly, and professional maintenance personnel should be equipped to take charge of the normal operation and maintenance of laminar airflow operating rooms.