In the production of 2nd generation ethanol, using Saccharomyces cerevisiae, the highest productivity obtained using C5/C6 fermenting yeast is in the co-fermentation phase, in which xylose and glucose are fermented simultaneously. Extending this phase in a fed-batch process increases the yield, rate and additionally reduces needed yeast amount for pitching. Extending this phase, as long as possible, would further enhance yield and economy of the process. To realise the concept a fermentation monitoring technique was developed and applied. Based on online measured refractive index an optimal residual sugar concentration could be maintained in the primary fermentor during the feed phase, requiring little knowledge of the nature of the substrate. The system was able to run stably for at least five fermentor volumes giving an ethanol yield >90% throughout the run. This was achieved with addition of only urea to the wheat straw hydrolysate and with an initial yeast pitch of 0.2 g/L total of finished broth. It has the potential to improve the fermentation technology used in fuel ethanol plants, which could help to meet the growing demand for more sustainable fuels.