Abstract: Prokaryotic expressed selenium-binding protein (SBP) of whitetip reef shark was identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) with high confidence. One of the peptides of SBP was judged as the self-hydrolysis peak of trypsin and mixture spectra were the reasons that they can not be selected for MS/MS analysis or can not be matched by database searching. In addition, it was found that despite the weak signal intensity of some lysine-terminated peptides in MALDI-TOF spectrum, MALDI-TOF/TOF spectra of them can provide nearly complete sequence information even can be used for de novo sequencing. Taking into account the above factors, the identification coverage of selenium binding protein increased from 35% to 51%.