This study examined the function of β-1,3-glucanase in Brassica rapa with low temperature stress, whose encoding protein was identified by protein mass spectrometry analysis. The cDNA sequence of β-1,3-glucanase was cloned by RT-PCR of winter rapeseed from Longyou6 and Tianyou4 cultivars. Semi-quantitative PCR and real-time fluorescence quantitative PCR were used to study the expression pattern of β-1,3-glucanase in response to low temperature stress. The open reading frame of the β-1,3-glucanase gene was obtained at a length of 1 032 bp, and encoded 343 amino acid. The molecular weight was 38.102 kDa and the isoelectric point 6.63, with 93.94% amino sequence similarity to B. rapa subsp. chinensis and B. napus. The protein encoded by this gene is a hydrophilic protein with one signal peptide and two transmembrane domains. The prediction of the secondary structures indicated that β-1,3-glucanase is a protein with more α-helices. The predicted β-1,3-glucanase contains a conserved amino acid sequence corresponding to the plant Glycol-Hydro 17 superfamily. RT-PCR and semi-quantitative RT-PCR results showed that β-1,3-glucanase was upregulated in response to 4°C, and the gene was upregulated to peak in expression at -4°C; However, the expression was inhibited at lower temperature (-8°C). The β-1,3-glucanase gene cloned from the winter B. rapa cv. ‘Longyou6’ might play a role in cold tolerance.