The impact of composition, concentration, and pH of buffer solution on the activity and viability of Escherichia coli BB cells has been studied. Potassium/sodium phosphates with different composition and pH and TRIS buffer solutions used for microbiological and biotechnological applications as well as in microbial bioanodes were compared. The optimal buffer solution to achieve the highest dehydrogenase activity was 50 мМ potassium phosphate buffer solution, KH2PO4, isotonized by sodium chloride either with pH 7.4 for freshly obtained E. coli cells or with pH 7.8 to maintain the activity and viability of the microorganisms while storing. The cells concentration in solution was important to achieve the highest dehydrogenase activity, viz. 0.6–0.8 mg of cells (in terms of the dry weight) per mL of buffer solution with 0.2 M dextrose (substrate) was optimal. The good correlation between dehydrogenase activity and electrochemical current responses of bioanodes with E. coli and methyl orange as a redox mediator was shown.