An online Fourier Transform Infrared Spectrometer and an ATR (Attenuated Total Reflection) probe,specifically at the application of real time measurement of the reaction substrate concentration in biological processes,were designed.(1) The spectrometer combined the theories of double cube-corner reflectors and flat mirror,which created a kind of high performance interferometer system.The light path folding way was utilized to makes the interferometer compact structure.Adopting double cube-corner reflectors,greatly reduces the influence of factors in the process of moving mirror movement such as rotation,tilt,etc.The parallelogram oscillation flexible support device was utilized to support the moving mirror moves.It cancelled the friction and vibration during mirror moving,and ensures the smooth operation.The ZnSe splitter significantly improved the hardware reliability in high moisture environment.The method of 60° entrance to light splitter improves the luminous flux.(2)An ATR in situ measuring probe with simple structure,large-flux,economical and practical character was designed in this article.The transmission of incident light and the light output utilized the infrared pipe with large diameter and innerplanted-high plating membrane,which conducted for the infrared transmission media of ATR probe.It greatly reduced the energy loss of infrared light after multiple reflection on the inner wall of the light pipe.Therefore,the ATR probe obtained high flux,improved the signal strength,which make the signal detected easily.Finally,the high sensitivity of MCT (Mercury Cadmium Telluride) detector was utilized to realize infrared interference signal collection,and improved the data quality of detection.The test results showed that the system yields the advantages of perfect moisture-proof performance,luminous flux,online measurement,etc.The designed online Fourier infrared spectrometer can real-time measured common reactant substrates such as glycerin in the bioreactor under high quality.There is a large potential market,such as online analysis in biological,chemical testing and material analysis.