As an important freshwater aquaculture species in China, Carassius auratus culture is currently suffering from serious losses due to infectious diseases. The use of antibiotics in preventing and controlling the bacterial diseases has caused the evolution of resistant strains of aquatic pathogens. This is a serious constraint on aquaculture development. Therefore, some alternative measures need to be developed to improve the quality and sustainability of aquaculture production. Probiotics can be a substitute for antibiotics. Lactobacillus sp. from the host or the culture environment have shown to have ability to better adapt to the culture environment and colonize in the host gut, which help them exhibit their probiotic effects better. This study intends to screen probiotic candidates from C. auratus according to their in vitro probiotic properties, which include acid and bile tolerance ability, antimicrobial activity, cell surface hydrophobicity, aggregation ability, in vitro adhesive ability and pathogenicity in crucian carp. Based on biochemical tests and 16S rRNA gene sequence analysis, thirty-eight strains of Lactobacillus sp. were isolated from the gut of C. auratus, which belonged to Lactococcus, Leuconostos, Carnobacterium and Enterococcus faecium, respectively, and eight of them were selected to evaluate the in vitro probiotic properties. The results showed that all eight strains of Lactobacillus sp. bacteria could tolerate acid (pH 4.6) and bile (10% bile of C. auratus), and exhibit good antibacterial activities against four pathogens including Aeromonas hydrophila, A. caviae, Streptococcus agalactiae and Vibrio parahaemolyticus. Five of eight strains showed high cell surface properties, i.e. hydrophobicity (63%-89%) and autoaggregation (37%-45%), and their adhesive rates to C. auratus intestinal mucus were ranged from 4.5% to 7.9%. These five isolates differed in coaggregation ability, but exhibited good adhesion inhibition rates for the binding of A. hydrophila to C. auratus intestinal mucus, with the inhibition rates of 30% to 35%. Three L. lactis isolates 16-7, 16-19 and 16-24 with a higher adhesive ability showed no pathogenicity when administered by intraperitoneal injection to C. auratus. Based on our data, the three L. lactis isolates could be considered as potential probiotic candidates for C. auratus farming. Further in vivo evaluation needs to be performed in the future study.