A novel on-line monitoring approach implementing an improved sampling methodology is presented here. A sample is directly withdrawn from the bioreactor without a need for a filtration or a dialysis probe, extensively diluted in a flow-through fashion and mixed with a mobile phase consisting of a buffer and a soluble mediator. The developed sampling system efficiently controls dilution of the sample prior to biosensor assay covering full dynamic range of glycerol concentrations during its fermentation to 1,3-propanediol. The new sampling protocol minimizes problems associated with the use of traditional on-line sampling methods implementing membrane separation or filtration techniques. Concentration of glycerol in the sample is determined by a flow injection analysis (FIA) with an amperometric biosensor using glycerol dehydrogenase and diaphorase with a throughput of nine samples per hour. The biosensor performance was validated by a reference analytical method (HPLC) with a very good agreement. (C) 2008 Elsevier Inc. All rights reserved.