Abstract: The technique of homologous cloning and Rapid Amplification of cDNA Ends(RACE) was used to amplify full length cDNA of immunoglobulin light chain 3 isotype (IgL3) gene from largefin longbarbel catfish (Mystus macropterus Bleeker). IgL3 in M.macropterus has 947 nucleotides, including 5￠-UTR of 58 nucleotides, 3￠-UTR of 184 nucleotides and an open reading frame with 705 nucleotides encoding a peptide of 234 amino acids. The deduced amino acid sequence contains an constant region(CL) and a variable domain(VL) consisting of 4 frame regions(FRs) and 3 complementary determining reg-ions(CDRs). The IgL3 comparison in seven teleost species showed that IgL3 in M.macropterus shared the highest identity (68.8%) with that(F) in Ictalurus punctatus, and the lowest identity(47.2%) with that in Salmo salar. Phylogenetic tree based on some teleost IgL amino acids showed that IgL3 in M.macropterus was clustered closely with that (F) of I. punctatus and grouped with IgL3 in other teleost, and was far away from L1 and L2 of teleost. Real-time PCR showed that IgL3 mRNA expression of M.?macropterus was mainly detected in head kidney, spleen and blood cells and increased significantly in these tissues after injection of Aeromonas hydrophila. The result indicated that head kidney, spleen and blood cell are main organs for IgL3 expression after stimulation, and play a critical role in immunity interaction in M.?macropterus.