The extractive electrospray ionization (EESI) technique is an emerging soft ionization technique and it can be used for the sensitive detection of large molecules and small molecules in solid, liquid, gas and viscous samples without sample pretreatment. Based on the brief introduction of the mechanism of EESI, its applications on the protein analysis were reviewed in detail. Compared to the commercial electrospray ionization (ESI) source, EESI maintains the native structure of protein and thus the biological activity of the protein to the most extent, which shows it can be potentially applied in the fields of preparation of protein microarray, high resolution hydrogen/deuterium exchange mass spectrometry (structure analysis of protein), and metrology for protein.