As one of the important problems in global change, elevated ultraviolet-B (UV-B) radiation induced by the depletion of stratospheric ozone layer has received more and more attentions around the world. Field experiment with barley was conducted to investigate the effects of elevated UV-B radiation on microbial biomass carbon and nitrogen in rhizosphere and nonrhizosphere soil. The experiment was designed with two UV-B radiation levels, i.e., elevated (E, simulating 25% stratospheric ozone depletion) and ambient (A, control), and performed at the Station of Agricultural Meteorology, Nanjing University of Information Science and Technology, Nanjing, China. Compared with the control, elevated UV-B radiation significantly depressed shoot biomass by 13.2–42.6% and root biomass by approximately 50% from jointing to ripening stage. Elevated UV-B radiation significantly increased microbial biomass C and N in nonrhizosphere soil in most cases, but significantly decreased microbial biomass C and N in rhizosphere soil. Further researches are needed to elucidate whether the above findings are connected with the changes in composition and amount of root exudates induced by elevated UV-B radiation, which can mainly affect the dynamics of soil microbial biomass.