Plant biomass is a key parameter for estimating terrestrial ecosystem carbon (C) stocks, which varies greatly as a result of specific environmental conditions. Here, we tested environmental driving factors affecting plant biomass in natural grassland in the Loess Plateau, China. We found that above-ground biomass (AGB) and below ground biomass (BGB) had a similar change trend in the order of Stipa bungeana > Leymus secalinus > Artemisia sacrorum > Artemisia scoparia, whereas shoot ratio (R/S) displayed an opposite change trend. There was a significantly positive linear relationship between the AGB and BGB, regardless of plant species (p < 0.05). Furthermore, more than 50% of the AGB were found in 20-50 cm of plant height in Compositae plants (A. sacrorum, A. scoparia), whereas over 60% of the AGB were found in 20-80 cm of plant height in Gramineae plants (S. bungeana, L. secalinus). For each plant species, more than 75% of the BGB was distributed in 0-10 cm soil depth, and 20% was distributed in 10-20 cm soil depth, while less than 5% was distributed in 20-40 cm soil depth. Further, AGB and BGB were highly affected by environmental driving factors (soil properties, plant traits, topographic properties), which were identified by the structural equation model (SEM) and the generalized additive models (GAMs). In addition, AGB was directly affected by plant traits, and BGB was directly affected by soil properties, and soil properties associated with plant traits that affected AGB and BGB through interactive effects were 9.12% and 3.59%, respectively. However, topographic properties had a weak influence on ABG and BGB (as revealed by the lowest total pathway effect). Besides, soil organic carbon (SOC), soil microbial biomass carbon (MBC), and plant height had a higher relative contribution to AGB and BGB. Our results indicate that environmental driving factors affect plant biomass in natural grassland in the Loess Plateau.