Objective To explore the effect of one-to-one practical training and multi-dimensional assessment for cleaning staff on the disinfection efficacy of bed units.Methods Two rounds of one-to-one practical training were conducted on 10 cleaning staff from 5 wards with prospective research method. Multiple indicators were adopted to assess the process and efficacy of disinfection on bed units in three rounds (before training, after the first training, and after the second training).Results Compared with before practical training, natural bacteria killing rate increased from 51.95% to 91.85% (P < 0.05) after one-to-one practical training. The percentage of decreased ATP detection value increased from 45.00% to 82.50% (P < 0.05). The fluorescence labeling clearance rate increased from 55.00% to 85.00% (P < 0.05). The implementation rate of disinfection specification increased from 62.00% to 92.00% (P < 0.05).Conclusion Carrying out one-to-one practical training and assessment for cleaning staff can significantly improve the disinfection efficacy of bed units and reduce the risk of healthcare-associated infection. The ward cleaning staff should conduct at least one-to-one practical training once to ensure the disinfection efficacy before taking office.