A method for studying of sorbitol dehydrogenase activity of Acetobacter suboxydans has been worked out which can be applied to other microbe cells too. The enzyme transformation of D-sorbitol into L-sorbose is determined through the reaction between ketohexose and 2-thiobarbituric acid under heating in 1 M oxalic acid (pH = 0.64) medium for 15 or 30 min. The yellow-coloured product was measured at 400 nm. The method is characterized with its high specificity, good reproducibility and accuracy, which allows for its application for quantitative analysis of sorbitol in dietetic foods. For this purpose a sorbitol dehydrogenase preparation isolated from bacterial cells is needed. The main purpose of the spectrophotometric method is to use it as a control in the process of microbial transformation of sorbitol into sorbose both under laboratory and industrial conditions.