Glutathione reductase is one of enzymes participating in the oxidation reduction reaction in organisms, which plays an important role in the process of stress tolerance. As a kind of cyanobacteria, Nostoc flagelliforme possesses strongly resistance to environment stresses，so it is meaningful to study the role of glutathione reductase in the resistant mechanism. In order to explore the biological functions of glutathione reductase in process of stress tolerance in N. flagelliforme, we cloned the complete open reading frame of the gene NfGR, and took a bioinformatics analysis of gene sequence. The ORF of NfGR contains 1 374 bp, encoding 458 amino acids, and its relative molecular weight and theoretical isoelectric point is 49.42 kD and 5.49, respectively. Amino acid sequence analysis showed that the protein NfGR has two domains of NADBRossmann superfamily and Pyr redox dim superfamily, and amino acid sequence of NfGR protein is similar to that of Nostoc punctiforme with similarity up to 93%. Phylogenetic tree reconstructed by neighbor joining method clearly showed that NfGR protein was classified into a subgroup with N. punctiforme, which suggested their close relationship. qRTPCR result showed that the expressions of NfGR gene were upregulated under different concentrations of PEG6000, and reached peak at 8% PEG6000 concentration, with the relative expression level of 32.69. It is hypothesized that glutathione reductase may be involved in the response to stress resistance of N. flagelliforme according to its expression characteristics.