Corn and alfalfa intercropping is considered an important practice for controlling soil erosion. The effects of herbicide application during the emergence and growth periods of alfalfa have not yet been studied. Therefore, this study was conducted to determine the impacts of two herbicides (nicosulfuron and atrazine) on alfalfa at different growth stages (before emergence, at emergence, and during the branching stages). The results revealed that herbicide application to maize significantly decreased emergence, plant height, stem diameter, biomass and grain yield. The mortality rate of alfalfa during the first year’s branching stage was the lowest, indicating that tolerance to this herbicide improved. The herbicides had negative impacts in a dose-dependent manner; for example, the same dose effects gradually increased first but then decreased. Moreover, the root-to-seedling ratio, activity index, and contents of soluble sugars and soluble protein in alfalfa significantly decreased in all three treatment groups, whereas the activities of three antioxidant enzymes significantly increased. The residual effect of the herbicide in the field completely disappeared when the interval between applications exceeded eleven months. Therefore, these findings indicate that herbicide application has negative effects on alfalfa growth.