For the development of a bio-sensor based on fluorescence excitation in transmission, a plastic substrate with excellent optical characteristics, such as high optical transmission in the visible range and low self-fluorescence, is required. Polyethylene (PE) films have been found to meet these optical characteristics. However, the adhesion of short sequences of oligonucleotides used as bio-probes, which are needed to develop a genosensor, has not proven ideal on this substrate, due to its olefinic composition. In order to overcome this challenge, a physical-chemical surface modification of PE films has been performed using a corona treatment, followed by the attachment of functional organic compounds. The surface modified substrates have been characterized for their wettability, surface energy, zeta potential and surface topography. The bio-probes’ adhesion on the treated surfaces has been tested by monitoring the fluorescence of dye-conjugated complementary sequences of the oligonucleotides deposited on the modified PE substrates. Finally, two modified PE substrates, comprising the best adhesion of the bio-probes, have been used to prepare a genosensor for the detection of the DNA of the insect Hermetia illucens. As expected, the better adhesion of bio-probes on the functionalized PE substrate has allowed a significant improvement of the bio-sensor’s limit of detection (LOD).