A mid-infrared (MIR) sensor using the attenuated total reflection (ATR) technique has been developed for real-time monitoring in biotechnology. The MIR-ATR sensor consists of an IR emitter as light source, a zinc selenide ATR prism as boundary to the process, and four thermopile detectors, each equipped with an optical bandpass filter. The suitability of the sensor for practical application was tested during aerobic batch-fermentations of Saccharomyces cerevisiae by simultaneous monitoring of glucose and ethanol. The performance of the sensor was compared to a commercial Fourier transform mid-infrared (FT-MIR) spectrometer by on-line measurements in a bypass loop. Sensor and spectrometer were calibrated by multiple linear regression (MLR) in order to link the measured absorbance in the transmission ranges of the four optical sensor channels to the analyte concentrations. For reference analysis, high-performance liquid chromatography (HPLC) was applied. Process monitoring using the sensor yielded in standard errors of prediction (SEP) of 6.15 g/L and 1.36 g/L for glucose and ethanol. In the case of the FT-MIR spectrometer the corresponding SEP values were 4.34 and 0.61 g/L, respectively. The advantages of optical multi-channel mid-infrared sensors in comparison to FT-MIR spectrometer setups are the compactness, easy process implementation and lower price. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.