This study evaluated the prebiotic effects of dietary inulin and Jerusalem artichoke tuber (JA) on juvenile Nile tilapia (Oreochromis niloticus). Five dietary treatments (each diet in four replicates) were formulated to incorporate inulin at 0 (control), 2.5 and 5 g kg−1 and JA at 5 and 10 g kg−1. Fish were reared in concrete ponds for 8 weeks. Fish fed the inulin diets exhibited better growth performance than fish fed the control diet, and fish fed the JA diets had the best growth performances among all diets tested. Dietary inulin and JA increased red blood cell number. Among the fourteen blood chemicals examined, dietary inulin or JA led to increased glucose, albumin, protein, magnesium, calcium, and iron content (P < 0.05). Inulin supplementation at 5 g kg−1 improved lysozyme activity and alternative complement haemolytic 50 (ACH50) activity. Dietary JA increased total immunoglobulin content, lysozyme activity, and ACH50 activity. Dietary inulin or JA increased the height of intestinal villi and goblet cell number. These findings indicate that inulin at 5 g kg−1 had beneficial prebiotic effects on juvenile Nile tilapia and that direct supplementation with JA at 10 g kg−1 had positive effects on growth and health. Thus, both inulin and JA have great potential for use as prebiotics in fish feed.