UV-B stress has become one of the main abiotic stress factors threatening crop growth and development, and miR408 plays an important role in plant abiotic stress response. MiR408 is a type of endogenous non-coding small fragment single-stranded RNA, which is mainly found in eukaryotes and participates in the regulation of plant growth and development by responding to various stresses in a manner that negatively regulates the target gene. The effects of photosynthesis and expression levels of miR408-OE rice plants and knockout UCL8 plants under UV-B stress were investigated. The results showed that under UV-B enhanced radiation, the chlorophyll content, intercellular carbon dioxide concentration, net photosynthetic rate and stomatal conductance of the overexpressed miR408 plants were significantly reduced. Under UV-B enhanced radiation, chlorophyll fluorescence kinetic parameters such as Fv/Fm and qP overexpressing miR408 plants were reduced. UV-B significantly inhibited the activity of SOD, POD and CAT in overexpressing miR408 plants and significantly increased their H2O2 and MDA contents. Under UV-B stress treatment, the yield traits such as the number of grains, the number of tillers and the quality of dry matter of overexpressing miR408 plants were significantly reduced. Therefore, UV-B enhances the photosynthetic rate, photosynthetic performance and disruption of the antioxidant system balance of overexpressing miR408 plants, which leads to a decrease in yield. It is speculated that overexpression of miR408 reduced the tolerance of rice to UV-B radiation, that is miR408 may play a negative regulatory role in response to UV-B stress.