Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is an emerging tool for detecting microorganisms. It can be used not only for rapid identification of microorganisms, but also for the research of resistance mechanisms. Producing β-lactamase is the main mechanism for resistance of β-lactam antibiotics in Gram-negative bacilli. Currently using MALDI-TOF MS for rapid detection of bacterial β-lactamase has been widely reported, including detection of β-lactam hydrolysis activity of β-lactamase, direct detection of β-lactamase molecule, peptide and relevant proteins, and detection of the single nucleotide polymorphisms (SNPs) of β-lactamase gene. This paper reviews current application of MALDI-TOF MS to the detection of β-lactamase based on the latest research findings.