Ambient ionization mass spectrometry(AI-MS) is a kind of novel MS technique,which is performed under ambient conditions without or with few sample pretreatment procedures.In recent years,the development and application of AI-MS technique have attracted increasing attention owing to its advantages,such as real-time,simplicity,rapidness and high throughput.The technical classification and principle characteristics of AI-MS and its application in microbiology research are summarized in this review,and the applications of AI-MS in the identification and typing of bacterial species,bacterial metabolite analysis and biofilm analysis,mass spectrometry imaging,etc.are mainly introduced.So far,ambient ionization combined with portable MS has been more and more widely used,which is anticipated to become a new hot spot in the field of mass spectrometry.