This paper aims to elucidate the effects of exogenous brassinolide (BL) on maize germination and seedling growth under chilling stress. The cold-resistant maize hybrid Tiannong 9 and the cold-sensitive hybrid Tianhe 1 were soaked at the germination stage (6 °C) and leaves were sprayed at seedling stage (4 °C), with BL at concentrations of 0, 0.01, 0.1, and 1 mg/L. The germination rate of the maize seeds and the changes in seedling biomass, antioxidant, photosynthetic, and plant endogenous hormone systems and chloroplast ultrastructures were determined. The results showed that the optimum concentration of BL to alleviate chilling stress in maize seedlings was 0.1 mg/L. This rate effectively increased the germination rate and plant biomass of maize and significantly increased the superoxide dismutase (SOD) peroxidase (POD) and catalase (CAT) activities, the net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (Tr), and seedling auxin (IAA), gibberellin (GA3) and trans zeatin nucleoside (t-ZR) contents under chilling stress. In addition, BL significantly reduced the malondialdehyde (MDA) content, abscisic acid (ABA) content, and intercellular carbon dioxide concentration (Ci). In the comparison of mesophyll cells, the chloroplast membrane of the treatment group was tightly attached to the stroma, and some of the plasma membranes were dissolved, but the overall structure of the chloroplast was relatively complete, and the osmiophilic granules were relatively few. The exogenous application of BL can effectively alleviate the damage caused by a low temperature in maize, maintain the normal characteristics of seedlings in chilling environments, and ensure the development and growth of plant tissue in the later stage.