Technical Abstract: Oligosaccharides feature high biological activity ensuring their wide application in the biotechnology, food, and cosmetic industries. On the other hand they are considered environmental pollutants. The study outlines a biosensor approach to detect these substances which is important from above standpoints. We examined substrate specificity of some yeast and bacterial strains to di- and trisaccharides, namely: maltose, cellobiose, sucrose, raffinose, lactose, and melibiose. The majority of strains were found to be sensitive to three substrates - maltose, cellobiose and melibiose. The profile of substrate specificity allowed singling out two strains having high oxidizing activity to sucrose and maltose. Thus, a strain of B. subtilis was active in oxidizing sucrose and maltose, while E. coli strain K-802 oxidized maltose with a high rate and had low oxidizing activity towards sucrose. These strains were chosen as a basis for biosensors to explore further their selective detection potential. The linear range of detection of maltose using B. subtilis was 0.005 - 0.5 mM; for sucrose, this range was 0.05 - 5 mM. The linear response range for the E. coli K-802 sensor (for maltose) was 0.05-5 mM. The variance was 6%. The time to measure a single sample was under 10-15 minutes.