Glycerokinase from Cellulomonas sp. was used to develop biosensor based on flow calorimetry for quantitative analysis of glycerol during bioconversion process. An automatic flow injection analysis device with the glycerol biosensor was built and tested during growth on glycerol of 1,3-propanediol-producing bacteria. The biosensor exhibited an extreme storage and operational stability enabling us to use it for more than 2 years without significant loss of sensitivity. No interference with 1,3-propanediol and fermentation medium was observed. The linear range of glycerol concentration up to 70 mM was extended by developed automatic dilution technique with the aim of automatic online monitoring of microbial process. The analytical system was able to monitor the bioconversion process in a fully automatic way during the whole run with sampling frequency of one sample per 10 min.