The role of volume hydrogel holographic gratings as optical transducers in sensor devices for point-of-care applications is increasing due to their ability to be functionalized for achieving enhanced selectivity. The first step in the development of these transducers is the optimization of the holographic recording process. The optimization aims at achieving gratings with reproducible diffraction efficiency, which remains stable after reiterative washings, typically required when working with analytes of a biological nature or several step tests. The recording process of volume phase transmission gratings within Acrylamide/Propargyl Acrylate hydrogel layers reported in this work was successfully performed, and the obtained diffraction gratings were optically characterized. Unslanted volume transmission gratings were recorded in the hydrogel layers diffraction efficiencies; up to 80% were achieved. Additionally, the recorded gratings demonstrated stability in water after multiple washing steps. The hydrogels, after functionalization with oligonucleotide probes, yields a specific hybridization response, recognizing the complementary strand as demonstrated by fluorescence. Analyte-sensitive hydrogel layers with holographic structures are a promising candidate for the next generation of in vitro diagnostic tests.