Ambient mass spectrometry is of increasing interest due to its remarkable features for direct analysis of complex samples with high sensitivity, high specificity and high throughput. Actual samples, even raw biological samples, can be directly analyzed with no or minimal sample pretreatment by using the advanced ambient ionization techniques. In this tutorial study, the fundamental working modes, the principles of ambient ion production, instrumentation and applications of typical ambient ionization techniques have been reviewed, giving emphases on the theoretical modes illustrating the working mechanism of various ionization techniques. The important progresses in food analysis, pharmaceutical analysis, environmental analysis, in vivo analysis, metabonomics studies, proteomics studies and ambient MS imaging have been systematically discussed, which show a clear trend that the development of ambient MS has being accelerated by the urgent practical need to detect trace compounds in matrices with high analysis speed. Moreover, the perspectives of these techniques have been exemplified using cases demonstrated in the literature.