In this work, effects of ozone treatment on the inactivation of microorganisms and physicochemical changes in wheat flour during the storage were evaluated. Total plate count (TPC) in wheat flour was found to be slightly reduced immediately after ozone treatment, but then significantly (P<0.05) decreased in the first few days' of storage. Ozone had more impact on pH value and a w than fatty acid value and water content of wheat flour. PPO activity in ozone treated flour was significantly inhibited and got slightly lower during storage. Ozone treated flour presented reduced pasting temperature and increased peak viscosity during storage, and no obvious changes were detected in the major volatile compounds as shown by the results of GC–MS. In addition, fresh noodles were made to evaluate the shelf-life extending effect as induced by ozone and noodles made from treated flour with 4days' storage presented a better microbial and color stability.This paper presents a new discovery of the non-immediately lethal effect of moderate ozone treatment on the microorganisms in wheat flour, which also indicates a potential for ozone as the alternative to chemical oxidants in flour and eliminates the potential residue of hazardous chemicals. Some physicochemical properties and the sensory qualities can be also improved. The discovery of this phenomenon would provide a new concept for “low-bacteria wheat flour” production and have important consequences for the application of ozone in food industry.