In order to comprehensively evaluate the effect of Lactobacillus rhamnosus in the culture of Litopenaeus vannamei and the appropriate addition form and concentration, seven experimental diets were formulated for L. vannamei (1.60±0.15 g): a control group (Group CK, basic diet); three live bacteria groups (LB1, LB2, LB3 supplemented with 1×106, 1×107, 1×108 CFU/g viable L. rhamnosus, respectively); and three heat-inactivated bacteria groups (DB1, DB2, DB3 supplemented with 1×106, 1×107, 1×108 CFU/g heat-inactivated L. rhamnosus, respectively). After a 28-day feeding trial, key findings revealed: (1) Compared to the CK group, the weight gain rates of the LB1, LB2, LB3, and DB2 groups increased by 5.35%, 14.40%, 9.78%, and 6.11%, respectively (P<0.05). (2) Intestinal amylase and lipase activities were markedly enhanced across all treatment groups (P<0.05). (3) The ratio of n-3 to n-6 polyunsaturated fatty acids in the muscles of the LB2 and DB2 groups exhibited a significant increase of 5.41% and 11.49%, respectively, compared with the CK group (P<0.05). Furthermore, the total content of PUFA in the DB2 group demonstrated a significant increase compared with the CK group (P<0.05). (4) All L. rhamnosus supplemented groups showed enhanced serum phenoloxidase and lysozyme activities relative to the control (P<0.05). Notably, heat-inactivated groups displayed increased total nitric oxide synthase activity (P<0.05). (5) Acid phosphatase activity in the hepatopancreas of shrimp in the LB2, DB2, and DB3 groups exhibited a significant increase of 31.51%, 28.44%, and 22.12%, respectively (P<0.05). While alkaline phosphatase activity increased in all groups except DB1 (P<0.05). (6) Histological analysis revealed improved hepatopancreatic health in treatment groups, characterized by organized cellular arrangement and distinctive stellate luminal structures in hepatic tubules. In conclusion, the dietary supplementation with L. rhamnosus significantly enhances growth performance, muscle quality, immune responses, and digestive enzyme activity in L. vannamei, while improving hepatopancreatic histomorphology. Optimal effects were achieved at 1×10? CFU/g for both viable and heat-inactivated formulations.