A new procedure for on-line monitoring of ethanol production in the wine fermentation bioprocess is presented. The method combines the use of a pervaporation unit inserted in a continuous flow system with the use of enzymic derivatisation and fluorimetric detection as a way of improving both selectivity and sensitivity. The derivatisation reaction was based on a two-step reaction involving alcohol oxidase (AOD) and horseradish peroxidase (POD) with fluorimetric detection at λ_ex = 310 nm and λ_em = 415 nm of the dimer formed. The efficiency of the derivatisation reaction was tested using different flow injection approaches with either solution phase or immobilised biocatalysts (in normal or stopped-flow modes in both cases). Finally, the approach based on the stopped-flow mode and AOD/POD immobilisation was selected for application to the continuous monitoring of ethanol production in an experimental laboratory built bioreactor prepared using baker’s yeast as active cells. The method shows different linear ranges depending on the situation of the catalyst and the mode used, with excellent precision (RSD 3.0–5.5%) and a sampling frequency of 5 h^–1. The use of this biosensing system was also tested by the standard addition method in the fermentation product with acceptable recoveries in all instances (90–105%). A fully automated approach to on-line bioprocess sample collection, dilution and monitoring of the fluorimetric product is also proposed.