There are still a large variety of microorganisms among aquatic animals, especially probiotic lactic acid bacteria in cold water fishes at high latitudes have not been fully developed. Hence, the present study aims to evaluate the probiotic potential of cold-adapted Lactobacillus strains isolated from the intestinal tract of cold water fishes (Xinjiang) and select candidates to be used as new food preservative agents and/or probiotic additives in feeding of aquaculture.A total of 43 Lactobacillus spp. were isolated from 16 kinds of intestinal tract of cold-water fishes. They were characterized by phenotypic methods, identified using Rep-PCR and 16S rRNA gene sequencing as four species: Lactobacillus sakei (22 isolates), Lactobacillus plantarum (16 isolates), Lactobacillus casei (4 isolates) and Lactobacillus paracasei (1 isolate). The in vitro tests included survival in low pH and bile, antimicrobial activity (against Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, Salmonella enterica subsp. enterica, Listeria monocytogenes, and Listeria innocua), resistance to 15 antibiotics and hemolytic tests. Among all 43 lactobacilli isolates, the 22 isolates showed a wide range of antimicrobial activity against 6 different pathogenic strains. There were twenty isolates growing at optimal temperature ranging 16~20 °C, which were initially considered to be cold-adapted strains. Two (2) Lb. sakei strains and 2 Lb. plantarum strains demonstrated the highest survivability after 4 h of exposure at pH 2.0. Most of the tested strains cannot be cultured after exposed into 0.5% bile solution for 4 h, while 2 Lb. plantarum strains (E-HLM-3, CQ-CGC-2) and 1 Lb. sakei strain M-DGM-2 survived even at 2% bile concentration. In addition, the safety assessment showed that 22 strains without any detectable hemolytic activity and resistant to glycopeptides (vancomycin, teicoplanin), levofloxacin, aztreonam, amikacin and oxacillin, while all the studied lactobacilli showed sensitivity to or semi-tolerant to other antibiotics.Based on all the experiments, 3 strains, including E-HLM-3, CQ-CGC-2, and M-DGM-2 might be a candidate of choice for using in the food preservative agents and/or probiotic additives in feeding of aquaculture.