One of significant characteristics of matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry ( MALD-TOF/TOF ) high-energy collision induced dissociation ( CID ) is to produce abundant immonium ( IM ) ions that can offer a wealth of information for peptide composition. However, MALDI-TOF/TOF is generally used for routine protein identification based on database search or de novo sequencing combined with chemical derivation. Consequently, the characteristics of IM ions may not be fully explored and utilized. Here, a total of 239 MS/MS spectra are used to explore the fragmentation features of IM ions with MALDI TOF/TOF spectrometry and their application for peptide identification. IM ion signals can be observed for 14 kinds of amino acids including histidine etc with a positive rate of more than 50%. We have found that the chemical nature of the amino acids and position effects are the two main factors that affect the intensity of fragment ions. In addition, false positive IM ions are mainly derived from Arg, Lys, Leu and Ile residues or mixture peptides. Besides the compositional information, partial sequence information can also be obtained by a comparison of the relative intensity of IM ions. These findings are helpful when performing manual interpretations and could be useful for improving current peptide search algorithms.