In recent years, the development of low-toxicity photopolymers for holographic recording in reflection mode has reached great importance. One of the main advantages of reflection holograms is that they can be reconstructed using white light, which has enabled that many researches have focused on the development of sensors for different types of analytes. Optical data storage and three-dimensional multiplexing of reflection holograms to improve the data storage density have also been investigated through reflection holograms. However, the photopolymers used in these researches have certain undesirable features such as the toxicity of some of their components and non-environmentally compatibility. The common used hydrophilic photopolymers content poly(vinyl alcohol) (PVA) or gelatine binder and monomers related to acrylamide. This compound has a high potential to cause cancer. For this reason, we developed a photopolymer called “Biophotopol” as a recording holographic material for optical applications. “Biophotopol” have a low toxicity, good recycling properties and is environmental-friendly. The basic formulation of “Biophotopol” includes an initiator system which is a free radical generator composed by triethanolamine as co-initiator and plasticizer and sodium salt 5’- 72 riboflavin monophosphate as sensitizer dye, sodium acrylate as polymerizable monomer and PVA as inert binder polymer. Additionally, a cross-linking agent, as N,N’-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) , can also be added. Volume transmission gratins and holographic lens have been fabricated in this photopolymer but not researches have been done to store reflection gratins. Is know that for higher spatial frequencies, the diffraction efficiency decreases considerably as the spatial frequency increases. In this sense, the general aim of this work has been fabricated reflection gratings in the symmetrical experimental setup in “Biophotopol” and to study the dependence of diffraction efficiency on physical thickness, recording intensity and exposure energy. First, films physical thickness was investigated. The maximum diffraction efficiency was obtained for thicknesses around 145 µm. The photopolymer layers uniformity was highly sensitive to drying and environmental conditions during the exposure stage. Therefore, both conditions were investigated and very controlled. An increase in diffraction efficiency was observed when the photopolymer films were cured with a LED lamp to improve the stability of the reflection holograms. The residual dye is eliminated during this process. The maximums diffraction efficiencies around 30 % were obtained for reflection gratings with a spatial frequency of 4738 lines/mm. The index modulation and optical thickness were obtained by fitting procedure through coupled wave theory. Experimental and theoretical results have been interpreted to modify the photopolymer formulation and exposure conditions in order to increase the diffraction efficiencies.