A kind of new ionization source, atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI), is introduced in the present paper. This ionization source was studied and developed on the basis of vacuum MALDI. It has been applied widely in the field of biomacromolecule. This ionization source has softer analyte ionization and produces fewer fragmentations compared with conventional vacuum MALDI. It works at atmospheric pressure, therefore eases to be coupled to mass spectrometers, acting as an external ion source, and greatly extends the application fields of MALDI. The principle, configuration and technical development of AP-MALDI are described detailedly. Moreover, the current applications and prospect of the device in analyzing biopolymers are summarized.