Water stress proteins play an important role in the process of drought tolerance in many plants. Nostoc flagelliforme showed strong tolerance to drought, salt and radiation stresses. In this study, using the genomic DNA of Nostoc flagelliforme as a template, the open reading frame (ORF) of a water stress protein gene was cloned by PCR amplification, and this gene was named NfwspA3 according to the online annotation via blastP in NCBI (https://www.ncbi.nlm.nih.gov/). Bioinformatic analysis showed that NfwspA3 gene contained 1017 base pair that encodes a putative protein consisted of 338 amino acid residues. The predicted isoelectric point was 4.31 and the theoretical molecular weight was 34.67 kDa. The encoded protein was predicted being a hydrophilic stable protein, and distributed in the cytoplasm. By analyzing the recombinant expression vector of pET32a-NfwspA3 under the condition of PEG-6000 simulating drought stress, it showed strong drought tolerance. Meanwhile, the recombinant expression strain showed higher activity of galactosidase. Thus, this work will provide a theoretical basis for detecting the molecular mechanism of water stress protein and a gene element that might be used for genetically modified breeding in Nostoc flagelliforme.