A real-time infrared (IR) spectroscopy measurement is an effective means to obtain enzymatic information either in vitro or in living cells,as it can provide direct,continue test of biomacromolecule reactions.The principles of measurements are performed basing on the fact that the absorption bands in spectra of reactants and products are usually separated to each other and changed independently with time.Therefore,it is possible to measure the enzymatic efficiency at any reaction time according to the changes of characteristic band,from either reactant or product.That is,IR spectroscopy can be used to obtain intracellular structural information during the cells metabolic processes,as which can provide detailed and reliable scientific evidences.In this paper,we summarized the new developments of IR spectra in the in vitro enzymatic assay for several representative enzymes,as well the screening of enzyme inhibitors,which was further extended to the identical aspect by using living cells as detection model.Such important enzymatic examination closes to the physiological conditions without labeling,supplying structural information of the related biomolecules.The developing trends of IR spectra are discussed and the perspectives of it in the research area are also provided in this review.