As one of the most promising analytical instruments, mass spectrometry(MS) technology has shown a broad application prospect in medicine, food, environment, human health, national security and other related fields. While the different types of analytes have diverse characteristics and largely add the difficulties of direct ionization and mass spectrometric analysis. Ambient mass spectrometry(AMS) is a kind of newly emerging technology performed under ambient conditions that allows the direct analysis of sample or sample surfaces with little or no sample pretreatment. The development and applications of ambient ionization mass spectrometry(AI-MS) that realized ionization under ambient conditions without sample pretreatment have become a frontier field in mass spectrometry and deserved much attention over the last few years. Ambient ionization has high sensitivity and specificity. It also can rapidly analysis real-time in situ and achieve high-throughput analysis without destructing the sample. As a soft ionization method, ESI offers unique advantages for proteomics by allowing the direct analysis of thermolabile compounds and forming multiply charged ions. The developments and applications of mass spectrometry imaging(MSI) have become a frontier field in mass spectrometry and molecular imaging. Some recent contributions on the development of ambient ionization can be applied to mass spectrometry imaging. This review focuses on the development of ionization mechanism, characteristics and applications of various ion sources, which are based on electrospray ionization (ESI) principle.