L-tryptophan (L-TRP) is an essential amino acid for the normal growth of crustaceans. As a nutritional supplement and antioxidant, L-TRP has the function of immune and antioxidant capacity regulation. From July to November, the effects of L-TRP on the immunity, antioxidant capacity and intestinal microflora of the Chinese mitten crab (Eriocheir sinensis) in pond culture were investigated. After feeding an L-TRP diet for 30 (named as August), 60 (named as September) and 106 (named as November) days, respectively, the activities of the immune and antioxidant enzymes in the hepatopancreas and hemolymph were evaluated, and the intestinal microbiota were profiled via high-throughput Illumina sequencing. The results showed that supplementation of L-TRP significantly increased the activities of AKP in the hepatopancreas in September, and significantly increased the activities of ACP in the hepatopancreas in August and September, and the hemolymph’s ACP activities also significantly increased in August and November (p < 0.05). Similarly, the activities of SOD, AOC and POD in the hepatopancreas significantly increased in September and November (p < 0.05) after feeding the L-TRP diet; meanwhile, the activities of SOD and AOC in the hemolymph also significantly increased in August (p < 0.05). However, in August, the L-TRP diet resulted in a significant increase in MDA activity in the hepatopancreas and hemolymph (p < 0.05). In addition, the results of the intestinal microbiota analysis showed that Firmicutes, Bacteroidetes and Proteobacteria were the dominant phyla in August, September and November, and Patescibacteria was the dominant phylum in September and November. After feeding the L-TRP diet, the richness of Cyanobacteria and Desulfobacterota significantly increased in August (p < 0.05), and the richness of Actinobacteriota significantly decreased in September (p < 0.05). Moreover, the L-TRP supplementation significantly reduced the abundance of ZOR0006 in the Firmicutes in September (p < 0.05). In conclusion, dietary L-TRP could improve the immunity and antioxidant ability and impact the intestinal health of E. sinensis at the early stage of pond culturing. However, long-term feeding of an L-TRP diet might have no positive impact on the activities of the immune, antioxidant enzymes and intestinal microbiota.