A Sequential Injection (SI) system was used to analyze the ethanol concentration in fermentation broth. The method is based on the use of immobilized NAD+dependent alcohol dehydrogenase. A non linear standard curve for ethanol (range 0.25–100 mM) was used to determine the concentration in fermentation broth and the results correlated well with HPLC measurements. The assay time was 140 s, 0.5 μmol of cofactor was used for each determination, and the relative standard deviation was less than 6% when analyzing fermentation samples. The assay system is very stable and makes it possible to reduce the cofactor consumption while keeping the system set up simple.