Background <p>Negative pressure wards are used to admit patients with respiratory infectious diseases. Most of the negative pressure wards are currently reconstructed, and the standard disinfection protocol has not yet been established.</p> Methods <p>In this study, we adopted the Delphi method to develop a disinfection protocol for negative pressure wards. First, we constructed a draft disinfection protocol based on literature analysis, theoretical analysis and investigation of the status quo. Then, we conducted two rounds of consultation through letters with fifteen experts in the management and disinfection of negative pressure wards from 6 provinces and cities around China.</p> Results <p>The final disinfection protocol of negative pressure wards contains 8 primary indicators, including air disinfection, surface disinfection, disinfection management of special hardware facilities, fabric disinfection, disinfection of secretions/excretions/vomitus, monitoring of disinfection effectiveness, management of cleaning and disinfection tools, and management of medical waste, along with 20 secondary indicators and 46 tertiary indicators.</p> Conclusions <p>The disinfection protocol for the negative pressure wards developed in this study is scientifically sound and highly operable, providing a reference for medical institutions equipped with negative pressure wards.</p>