This study investigated the influence of L-tryptophan (L-trp) on the survival, immune response and gut microbiota of the Chinese mitten crab, Eriocheir sinensis (with an average weight of 16.58 ± 2.20 g). After 30 days of feeding with diets supplemented with L-trp at 0.36%, 0.47%, 0.73% and 1.05% (groups 1, 2, 3 and 4, respectively), the survival rate and bacterial challenge (Aeromonas hydrophila) were evaluated, the activities of antioxidant and phosphatase enzymes in the serum were assessed, and the gut microbiota were measured via high-throughput Illumina sequencing. The results showed that the supplementation of L-trp significantly improved the survival rate of crabs (P < 0.05). After feeding for 7 days, it was observed that a high L-trp diet significantly increase the survival rate relative to a basal diet after a 96-h post-challenge with A. hydrophila (P < 0.05). The activity of CAT and AKP in the serum were increased by the addition of L-trp. The activity of CAT and AKP in the serum in group 4 were higher than those in group 1 (P < 0.05). Furthermore, we observed that adjunction of the L-trp can significantly increase the richness and diversity of the gut microbiota. The dominant phylum in the intestine of the Chinese mitten crab were Tenericutes, Proteobacteria, Firmicutes, Chloroflexi and Actinobacteria. The L-trp in the diets increased the richness of Proteobacteria, Firmicutes and Actinobacteria in the intestine significantly. These bacteria were all dominant bacteria and had a specific role in promoting the immunity of E. sinensis. Therefore, it could be inferred that L-trp supplementation is beneficial in the diet of E. sinensis. Based in these results, the dietary 0.47% or 0.73%L-trp supplemented is found to be optimum to improve E. sinensis survival.