With wolfberry (Lycium barbarum L.) as material, polymerase chain reaction (PCR) combined with RACE technology were used to clone a cDNA of WRKY from wolfberry, named as Lb WRKY₃. (GenBank accession No.KX196192). Meanwhile, on the basis of bioinformatic analysis, we performed the subcellular localization assays and tissuespecific expression analysis. The results indicated that: (1) Lb WRKY₃ has an open reading frame(ORF) of 1 068 bp, which encoded a protein of 356 amino acid residues. (2)Bioinformatic analysis indicated that Lb WRKY₃ protein contains the one conserved WRKY motifs, the predictive secondary structure showed that Lb WRKY₃ protein was made up of 15.82% alphahelix, 6.63% betaturn, 18.88% extended strand and 58.67% random coil. Lb WRKY₃ protein showed the highest homology identity with WRKY protein from Chrysanthemum x morifolium and Artemisia annua. (3)Subcellular localization assays showed that the Lb WRKY₃ protein was located in the nucleus. (4)Realtime PCR analysis indicated that Lb WRKY₃ was expressed in high transcript level in roots, low levels in flower. The Lb WRKY₃ gene expression could be detected during the whole period of fruit development. Interestingly, Lb WRKY₃ gene showed a high transcription level in 35 days. The expression level in the pulp was higher than that in the seed and peel . Lb WRKY₃ gene takes part in fruit development and sugar accumulation in wolfberry.