Abstract The location of air supply and exhaust, differential of supply and exhaust air, air change per hour and negative pressure have been proposed in the specification of negative pressured isolation ward. The purpose of negative pressure is to prevent virus from spreading out. The doorway size is an important factor on negative pressure. However, the doorway size is never suggested in the guidelines. The aim of this study is to investigate the effect of doorway size on pressure difference under different room sizes and air change rates. A volume adjustable chamber with independent ventilation system was used to simulate negative pressured isolation room without anteroom. By changing doorway size, the corresponding negative pressure could be measured under different room size and air change per hour (ACH). It was found that the negative pressure decreases with the increasing doorway size, and reaches to zero when the doorway size is large enough. When the doorway size is fixed, the pressure difference decreases with ACH. When the ACH is equal to 4, the pressure difference is always smaller than 8 Pa though the doorway size is kept at minimum (0.2cm). Therefore, to maintain negative pressure is equal to 8 Pa, the ACH is required to be greater than 4, or increasing the exhaust air at least 20% greater than supply air. The model of doorway size was also developed by using the experimental data. The results provide useful information for future design and improvement of negative pressured isolation ward.