Nitrogen (N) is the most limiting nutrient for maize, and appropriate N fertilization can promote maize growth and yield. The effect of N fertilizer rates and timings on morphology, antioxidant enzymes, and grain yield of maize (Zea mays L.) in the Loess Plateau of China was evaluated. The four N levels, i.e., 0 (N0), 100 (N1), 200 (N2), and 300 (N3) kg ha−1, were applied at two timings (T1, one-third N at sowing and two-thirds at the six-leaf stage of maize; T2, one-third applied at sowing, six-leaf stage, and eleven-leaf stage of maize). The results show that N2 and N3 significantly increased the plant height, stem and leaf dry weight, and leaf area index of maize compared with a non-N-fertilized control (N0). The net photosynthetic rate, transpiration rate, stomatal conductance, and leaf chlorophyll contents were lower, while the intercellular carbon dioxide concentration was higher for non-fertilized plants compared to fertilized plants. The activities of peroxidase (POD) and superoxide dismutase (SOD) increased with N rate, but the difference between 200 and 300 kg ha−1 was not significant; further, the isozyme bands of POD and SOD also changed with their activities. Compared with a non-N-fertilized control, N2 and N3 significantly increased grain yield by 2.76- and 3.11-fold in 2018, 2.74- and 2.80-fold in 2019, and 2.71- and 2.89-fold in 2020, and there was no significant difference between N2 and N3. N application timing only affected yield in 2018. In conclusion, 200 kg N ha−1 application increased yield through optimizing the antioxidant enzyme system, increasing photosynthetic capacity, and promoting dry matter accumulation. Further research is necessary to evaluate the response of more cultivars under more seasons to validate the results obtained.