The quality and quantity of different sugars play a very important role in studying the carbohydrate metabolism of yeast. During the bioprocesses there is a need to follow the concentrations of these sugars. Authors have reported on the development of biosensors for determination of glucose and maltose previously. The aim of this research was to construct a sensor for determining galactose in fermentation broths to prepare the basis for an online monitoring system. Using a modified thin-layer enzyme cell connected to an electrochemical detector cell, a biosensor has been developed for this purpose. Galactose was oxidized with immobilized galactose oxidase enzyme (EC 1.1.3.9) and the hydrogen peroxide generated during the enzyme reaction was determined with an amperometric detector. The parameters for the biochemical and electrochemical reactions were optimized. The pH optimum of 6.6 was found when using phosphate buffer. The buffer solution completed by micro elements (Mg2+, Se2+) gave more stable signs. The activities for raffinose, lactose, glycerol and dihydroxyacetone were 68, 16, 6 and 430%, respectively. With the thin-layer cell more than 900 samples were measured in 6 weeks. Samples obtained from different fermentations were measured with the newly developed galactose sensor and the results were compared with the standard UV method. The correlation coefficient was 0.991. The results showed that the application of the new biosensor was successful.