Abstract: In order to improve the resistance of tobacco seedlings to low temperature stress, the physiological and molecular mechanisms of oligosaccharides were studied. In this study, the low temperature sensitive tobacco variety "K326" was used as the test material, to study the effects of foliar spraying with different concentrations of chitosan oligosaccharides, brown alage oligosaccharides, and mannose oligosaccharides on the growth and physiological characteristics of tobacco seedlings under low temperature stress, and to explore the molecular mechanism of enhancing tobacco resistance to low temperature. The results showed that compared with the low temperature stress group, foliar spraying of different types and concentrations of oligosaccharides could significantly increase the maximum leaf area and biomass of tobacco seedlings by 22.45%-62.84%, increase root activity and the content of chlorophyll by 32.02%-131.72% and 10.02%-34.76%, respectively. The content of osmoregulatory substances was increased by more than 14.10%, significantly increased the activities of antioxidant enzymes, such as superoxide dismutase, peroxidase, catalase, and enhanced the activity of glutathione reductase, and increased the reduced glutathione content. Combined with the affiliation function analysis, it was concluded that tobacco seedlings with foliar spraying of 200 mg/L mannose oligosaccharides had higher resistance to low temperature stress than 500 mg/L mannose oligosaccharides and 200 mg/L chitosan oligosaccharides. Further transcriptome sequencing analysis showed that mannose oligosaccharides treatment significantly increased the expression of amino acid degradation, glycerolipid metabolism, redox enzyme activity and catalytic activity. This study demonstrated that foliar application of 200 mg/L mannose oligosaccharides significantly enhanced the cold stress tolerance of tobacco seedlings, outperforming both 500 mg/L mannose oligosaccharides and 200 mg/L chito oligosaccharide.