Early weaning stands as a critical phase in the breeding of juvenile largemouth bass (Micropterus salmoides), aiming to ascertain optimal size parameters and evaluate physiological and biochemical impacts. This study investigated the effects of early weaning on survival rates, antioxidant and digestive enzyme activities, and intestinal microbiota. Five length categories [(1.06±0.02) cm, (2.04±0.03) cm, (3.02±0.04) cm, (4.05±0.04) cm, (5.01±0.04) cm] of juvenile fish were subjected to early weaning experiments. Data on natural mortality, survival rates, and cannibalism rates were analyzed. Antioxidant enzyme activities in the liver and muscle, as well as digestive enzyme activities in the stomach, gut, and pyloric ceca, were assessed pre and post-early weaning. Intestinal microbiota analysis was conducted using high-throughput sequencing. Findings revealed: (1) Highest natural mortality (60.31%) and cannibalism rates (30.85%) occurred at 1 cm length level, with the lowest survival rate (8.84%). Conversely, the 4 cm and 5 cm length levels exhibited no natural mortality or cannibalism, with 100% survival. Increasing body length correlated with decreased natural mortality and cannibalism rates alongside heightened survival rates. (2) SOD activity in the liver and muscles increased by 86.97% and 16.62%, respectively (P＜0.05), post-early weaning, whereas CAT activity in the liver decreased by 58.60% (P＜0.05). T-AOC and MDA levels in the liver and muscles remained statistically unchanged. (3) Trypsin, lipase, and amylase activities were highest in the pyloric ceca, followed by the gut, and lowest in the stomach. Post-early weaning, trypsin, lipase, and amylase activities in the gut decreased significantly by 50.11%, 40.71%, and 42.79%, respectively. Trypsin activity in the stomach decreased by 18.33% (P＜0.05), with no significant changes observed in the pyloric ceca. (4) At the phylum level, Actinobacteria, Proteobacteria, Firmicutes, and Verrucomicrobiota were predominant in juvenile Micropterus salmoides guts. Actinobacteria decreased by 67.42% (P＜0.05), while Firmicutes increased by 360.44% (P＜0.05) post-early weaning. At the genus level, Mycobacterium, Microbacterium, Paracoccus, and Gammaproteobacteria were predominant pre-weaning, transitioning to Lactococcus, Mycoplasma, Bacillus, and Achromobacter post-weaning. LEfSe analysis identified 51 significantly altered bacterial groups in the gut post-early weaning. Findings suggest 3–4 cm length as the optimal early weaning size for juvenile Micropterus salmoides. Early weaning-induced stress leads to oxidative damage, affecting antioxidant enzyme activities. Digestive enzyme activities exhibit significant alterations during transfeeding, with a notable decline. Intestinal microbiota composition undergoes significant changes post-early weaning, with Firmicutes emerging as the predominant phylum. This study provides valuable insights for enhancing survival rates and digestive efficiency during the early weaning phase of juvenile Micropterus salmoides.