Directed evolution was performed for toluene-o-monooxygenase (TOM) and toluene/xylene-o-monooxygenase (ToMO) to obtain differential and improved reaction rates with respect to each chlorinated ethenes. Peroxide shunt was applied to remove the requirement for NADH, thus eliminating the need for complex NADH regeneration schemes for live cells on the biosensor tip and long-term application of enzyme biosensor would be possible. A three-layer sandwich structured sensing tip was constructed. The monooxygenases were immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel that incorporated fluoresceinamine (FLA), layered on an inner glass disk. The sensor operated in a static mode at room temperature and the intensity variation caused by hydrogen ion served as an analytical signal. Calibration curves were obtained for TCE and PCE, with concentration ranges 0.2-100 mg/l. The detection limits were 10μg/l for TCE and PCE. The method reproducibility was tested. The method was successfully applied to the detection and determination of these chlorinated ethenes in water samples, without sample preparation steps.