Ethylene is involved in regulating plant response to multiple stress. However, the physiological and molecular mechanisms by which ethylene regulates the response of early rice seedlings to low temperature stress remain unclear. In this study, three substrates including soilless substrate, fermentation substrate and nutritional soil were selected for raising early rice Zhongzao39 to the two-leaf stage. After being sprayed ethephon, rice seedlings were treated with low temperature for 3 days, and then the physiochemical properties and the expression of cold resistance genes were determined. Equal amount of water was sprayed as control. The results show that ethephon treatment significantly increases the growth of rice seedlings at low temperature, increases the contents of nutrients and osmotic regulation substances proline and soluble sugar, and enhances the antioxidant system enzyme activities and REDOX status (AsA/DHA ratio) of rice seedlings. The expression of cold tolerance genes such as OsCOIN, TERF2, OsLti6a and OsCDPK7 in seedlings are improved. These results suggest that ethylene spraying can enhance the resistance of rice seedlings to low temperature stress by regulating the content of osmotic substances, REDOX level and expression of cold tolerance genes in rice.