Robust and cost-effective stimuli-responsive polymers show prominent advantages to be integrated into detection devices. In addition, modified track-etched membranes with chemical-sensing polymers possess additional robustness features such as including the sensing material into sub-micron pores. In this study, we report the preparation of track-etched PET membranes with fluorescent response in direct relation to changes in the pH of the environment. Immobilised Fluorescein and Green Fluorescent Protein have been used as pH-sensor elements. The former was not sensitive to pH, while the latter had a similar pH sensitivity to that of the free protein. Modifications of track-etched membranes were carried out by grafting polymerisation initiated by the remnant radicals, a straightforward technique for selective modification of the inner wall of pores. The biosensor prepared with the fluorescent protein was able to sense the pH of a buffer solution in the range 4 to 8. Furthermore, this membrane evidenced capacity to sense the pH of the cell growth by in situ fluorescence intensity detection during E.coli cell culture in microwells.