An optical sensing membrane has been fabricated to measure the concentration of dissolved oxygen(DO) and pH value simultaneously. It has employed HPTS as a pH sensitive dye and a ruthenium(II) complex as a DO sensitive dye. The sensing membrane has been applied to wells in a 24-well microtiter plate. Using the 24-well microtiter plate the concentrations of dissolved oxygen and pH values have been on-line monitored during the cultivations of E.coli DH5${alpha}$, B.cereus 318 and P.pastoris X-33. On-line monitoring of DO and pH in microorganism cultivation processes showed good performance of the sensing membrane containing 5 mM HPTS and 2 or 5 mg/mL Rudpp.