Abstract: The application progress on biological mass spectrometric analysis of nucleotide has been reviewed in the past decade. The principle of electrospray ionization-mass spec-trometry (ESI-MS) and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) is described. The advantage of biological mass spectrometry with accuracy, high speed, high sensitivity is introduced. The study results on mass spectrometric analysis of single nucleotide polymorphisms genotyping, short tandem repeats, diagnosis of diseases suffered from gene defect and sequencing of oligonucleotide fragment. The ESI-MS determination of non-covalence complex composed of oligonucleotide is discussed, including study on advanced structure of oligonucleotide, complex of oligonucleotide with protein, interaction between oligonucleotide and other small molecules. Simultaneously, the issue is put forward and the development direction is prospected.