Abstract: Time-of-flight secondary ion mass spectrometry（TOF-SIMS）is becoming an important technique for chemical surface analysis since its instrumental performance has been kept improved. The major analytical functions of TOF-SIMS include surface mass spectrometry, chemical mapping and depth profiling. This paper as the second part of the review titled as “Applications of Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) in Biomaterials and Life Science” introduced the applications of TOF-SIMS imaging and depth profiling in biomaterials and life science. Three major applications were discussed: how TOF-SIMS imaging technique was used as an important diagnosis tool for bioarray fabrication, and TOF-SIMS imaging and depth profiling characterization of biomolecule distribution in cell and tissue. In addition, the following experimental aspects were briefly described: sample preparation at low temperature, matrix-enhanced SIMS technique, using cluster primary ion to increase molecular secondary ion yield, as well as depth profiling using C₆₀ ion source which bring lest damage to underlying materials during the sputtering process. Finally, an outlook for the application of TOF-SIMS in biomaterials and life science was given.