The extensive application of nanomaterials had caused a great environmental load in soil environment. Thus, clarifying the process of environmental migration is significant for environmental safety and human health. In this study, alfalfa was used as an experiment crop to determine the effects of different doses of ZnO NPs on the growth of alfalfa and the soil environment. Contrary to the results from exposure to high-concentration ZnO NPs, a low-concentration of ZnO NPs could promote the growth of alfalfa. The Zn concentration in the alfalfa tissue and exposure dose showed a significant positive correlation. N750 treatment significantly reduced the number of alfalfa nodules, and a noneffective nitrogen fixation area in the nodules was found. ZnO NPs caused the rupture and bending of the nodule cell wall and reduced the concentration of nitrate--nitrogen. ZnO particles could enter the tissues at root tip and nodule and aggregated into larger particles. High-dose ZnO NPs reduced the relative abundance and diversity of the soil microorganisms. Therefore, short-term and high-dose exposure to ZnO NPs would cause multiple toxic effects to the plant and soil environment.