Integrating PDMS channels and chips containing pre-functionalized biosensors into microfluidic devices with irreversible sealing has been challenging because the integration process requires use of an O2 plasma treatment that usually destroys the biosensors. In this study, we examined the usefulness of introducing a shadow mask into the process as a method of protecting the pre-functionalized biosensors. Single nanowire sensors were pre-functionalized with fluorescently labeled biomolecules and then subjected to O2 plasma with and without the shadow mask. Results from the two groups were then compared. Those sensors without a shadow mask were destroyed, giving the sensor an infinite resistance and reduced fluorescence intensity. In contrast, the sensors with the shadow mask were protected and exhibited little changes in the resistance and fluorescence intensity. Then two different nanowires, aptamers incorporated polypyrrole nanowire (entrapment) and antibodies immobilized polyaniline nanowire (surface covalent binding), were used to investigate their detection performance before and after the plasma treatment in the presence of shadow mask. The protected samples showed a good sensitivity to the targets with a slight reduction in response compared with the as-prepared samples. After the O2 plasma treatment, the microfluidic channels were integrated with single nanowire biosensors. This microfluidic biosensor showed a high sensitivity with about ~0.5 % change in conductance at the lowest IgE protein concentration of 10 pM.