This study investigates the kinetics of rhodamine B diffusion over a multilayer membrane composed of silk fibroin hydrogel and polyethylene terephthalate (PET) nonwoven fabric. To evaluate the impact of varying membrane structure thicknesses on dye diffusion rate, we used 2, 4, 8, 16, and 32 layers. The research indicated that the 32-layer membrane significantly slowed dye penetration. An initial linear release was seen until 2500 minutes, after which the rate of diffusion decelerated. Validation of the controlled diffusion behavior across the hydrogel layers was achieved by UV-Vis spectroscopic analysis, specifically targeting the absorbance peak at 554 nm of rhodamine B. The results of this study provide crucial insights for the advancement of hydrogel- based materials in sensor technologies, drug delivery approaches, and regulated dye release.