Herein, to improve the reflectivity of open-type retro-reflectors at 808-nm wavelength, the impact of glass micro-beads' refractive index on the retro-reflection is theoretically analyzed. The optical simulation software TracePro is used to simulate and analyze the performances of three retro-reflector's structural units (glass micro-beads) with different refractive indexes (1.93, 2.00, and 2.20). The theoretical reflectivity of the conventional open-type retro-reflector's structural unit on the ordinary substrate is only 23.6% when the light is perpendicularly incident, however its theoretical maximum reflectivity is enhanced by 3 times, reaching 98.1%, when a high-reflectivity substrate is used. A high-reflectivity optical film having a reflectivity of 99.3% is designed and fabricated at 808-nm wavelength, and a retro-reflector on the substrate coated with such high-reflectivity optical film is prepared. Retro-reflectors using these two types of substrates are experimentally evaluated, and the results demonstrate that the glass micro-beads with the refractive index of 1.93 on the high-reflectivity substrate provide the best retro-reflectivity. At vertical incidence, the retro-reflectivity value of the retro-reflector on the high-reflectivity substrate reaches 46.2%.