NK-LYSINs are small cationic antimicrobial peptides produced by cytotoxic T lymphocytes and natural killer cells. Antimicrobial peptides, the important components of innate immune systems, have attracted scientists' attention because they can be served as novel antimicrobials. Megalobrama amblycephala is an important freshwater fish cultured in our country. A serious disease, bacterial septicemia caused by Aeromonas hydrophila is a big threat to M. amblycephala, which might lead to a high mortality. In order to understand the possible function of NK-lysin in the M. amblycephala immune system, we amplified and characterized the nkla and nklb genes in M. amblycephala, which encode 122 and 136 amino acids respectively. Sequences analysis showed that NK-lysin belong to the saposin-like protein family, with 6 highly conserved cysteines and a SapB domain. In different healthy fish tissues, the M. amblycephala nkla was highly expressed in the spleen, lowly in the muscle and blood, While nklb had the highest expression in the intestine and the relative lower level in the head kidney. However, after A. hydrophila infection, the M. amblycephala nkla and nklb shared the same expression trend. In the head kidney, the mRNA level decreased remarkablely at 4 h. In the liver, the expression increased to the peak significantly at 4 h and then fell down to the normal level. However, in the spleen and intestine, the expression fell to the bottom at 12 h and then reached the peak at 72 h. These results suggest that NK-lysin might play a vital role in the M. amblycephala defense against A. hydrophlia infection. What's more, we constructed prokaryotic expression system and obtained the recombinant proteins of M. amblycephala nkla and nklb, which may provide information for NK-lysin's further functional study.