In most biophotonics applications of dye-gel blends, the gel is illuminated by a light source (usually a laser or LED) for a considerable period of time. Therefore, it is crucial to examine the temporal behavior of the corresponding luminescence features. In the present report, a photosensitive hydrogel was synthesized using Eosin Yellow (EY) as the dye, Polyvinyl Alcohol as the polymer and Borax as the cross-linking agent (EY-PVA-Borax hydrogel). Dynamic behavior of the corresponding fluorescence emission due to excitation of by a diode laser at λ = 445 nm (which is the isosbestic point of the EY absorption spectrum during the photodegradation process) were studied parametrically and evaluated using difference spectra analysis (DSA). In general, the wavelength at the fluorescence peak underwent hypsochromic shift with irradiation time, while the intensity increased in the first 9.0 s and then decreased gradually. Hydrogen bonding between the hydroxyl group of PVA and the carboxyl group of EY, aggregation of the EY molecules and debromination of the EY monomers under light irradiation were considered to explain the dynamic behavior of the fluorescence emission from the gel. Considering the use of hydrogels as photo-convertors, the influence of gel layer thickness on the fluorescence spectrum was examined. The results obtained are of particular importance for the design of fluorescence-based biosensors and also in the field of fluorescence biomodulation.