Constipation is a widespread gastrointestinal issue. This work aimed to investigate anti-constipation capacities of 9 novel strains of Lactobacillusand Bifidobacteriumusing in vitroexperiments for initial exploration and then further confirmed through in vivomodels including both zebrafish and mice. The in vitroexperimental results revealed that L. plantarumT34 and L. paracaseiYJ10 could survive under the simulated gastrointestinal tract conditions as standard strain L. rhamnosus(LGG) did. L. paracaseiYJ10 and L. caseiLc showed the highest 22 h's aggregation rate. L. rhamnosusW1 showed the highest coaggregation rate with E. coliand with S. aureus. T34 had the largest inhibition zones against S. typhimuriumand L. Monocytogenes.The highest adhesion rates with HT-29 cells were L. caseiLc, LGG and W1. Principal component analysis (PCA) and Fuzzy synthetic evaluation (FSE) were performed to evaluate the in vitroprobiotic potential of the 9 strains comprehensively. The order of scores on in vitroprobiotic property from high to low was as follows: T34, W1, YJ10, Lc, LGG, SLT, SS, Lb, YN4 and LQSQ. The subsequent in vivoexperiment of zebrafish further confirmed T34, W1 and YJ10 have better anti-constipation ability compared to other strains. Furthermore, T34 relieve loperamide-induced constipation by promoting gut transit in the mice model. In conclusion, T34 was demonstrated to prevent constipation and may be suggested for use in food and pharmaceutical goods.