This experiment was performed to identify the influence of dietary Saussurea lappa root (SLR) on the performance and general health status of Nile Tilapia fingerlings (O. niloticus). Four formulated diets with different SLR levels of 0.0, 2.5, 5 and 10 g/kg, respectively, were afforded to fingerling fish (15.42 ± 0.05 g) for 8 weeks. The feed efficiency ratio (FER), feed intake (FI) and feed conversion ratio varied with dietary SLR level in a linear model and a high feed efficiency rate was recorded at the 10 g/kg group, while FI and FCR exhibited an opposite trend (P < 0.001). Dietary SLR level influenced serum protein constituents, liver and renal function enzymes, triglycerides, cholesterol and glucose (P < 0.001). Serum Catalase (CAT), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) remarkedly increased with dietary SLR level and attained a level at 10 g/kg. Furthermore, serum lysozyme, complement C3 (C3), IgA and IgD were stimulated by 10 g/kg SLR. Intestinal digestive enzymes (lipase and amylase) increased with SLR level up to 10 g/kg. As the dietary SLR level raised, the cumulative survival percentage aginst A. hydrophila challenge increased and then reached a maximum at 10 g/kg SLR group. Moreover, gene expression of pro-inflammation cytokines (TNF-2a, IL-1β, and IL-10) in liver and kidney transcriptomes demonstrated effective immunostimulant capabilities of greater SLR inclusion levels in fish diet. Meanwhile, intestinal microbial investigation, revealed that high levels of SLR in tilapia fish feed significantly suppressed total bacterial count, and pathogenic bacterial count (such as, E. coli, Coliform, Aeromonas spp, Pseudomonas spp.), and stimulated lactic acid bacteria development. Finally, it is recommended to include a high level of SLR (5 or 10 g/kg) in the diet of O. niloticus fingerlings to enhance feed efficiency, antioxidant characteristics, and immunological response against bacterial infections.