Low temperature seriously affects the growth of crops and poses a huge threat to food production. The application of wood vinegar can be used as a cost-effective and environmentally friendly strategy to promote crop growth and enhance stress resistance, and the physiological resistance to low-temperature stress of rapeseed still needs further research. The present study investigated the effects of spraying wood vinegar on the growth, photosynthesis, osmotic adjustment, and antioxidant enzymes of rapeseed seedlings under low-temperature stress. The results showed that spraying wood vinegar at normal temperature reduced the stomatal conductance but increased the leaf area and total biomass of rapeseed and enhanced stomatal density and water use efficiency. The leaf area and total biomass of rapeseed sprayed with wood vinegar at low temperature improved by 22% and 31%, respectively, and stomatal density and water use efficiency increased by 14% and 83%, respectively, and intercellular CO₂ concentration and stomatal conductance were reduced by 9% and 41%, compared to the low-temperature, respectively. Besides, the application of wood vinegar liquid improved the proline, soluble protein, and soluble sugar content of leaves by 208%, 38%, and 115%, respectively, and the activity of superoxide dismutase increased by 27%, the content of malondialdehyde decreased by 46%, compared to the low-temperature. Spraying wood vinegar could alleviate low-temperature stress by improving the anti-oxidant enzyme content and osmoprotectants, reducing the stomatal conductance, and enhancing water use efficiency. These results provide new insights for wood vinegar to relieve the low-temperature stress of rapeseed, and this strategy can be used for low-temperature rapeseed cultivation and management, and benefit farmers’ plant profit.