Abstract: To determine the effects of dietary Bio100 level on growth performance, digestive enzyme activity, serum immune and biochemical indices of juvenile hybrid groupers (Epinephelus fuscoguttatus♀×E.lanceolatus♂), we prepared six experimental feeds with Bio100 contents of 0% (control), 0.05% (T1), 0.10% (T2), 0.15% (T3), 0.20% (T4) and 0.25% (T5). The fish (23.33±0.33) g were randomly assigned to six groups with three replicates per group and 20 fish per replicate, and were fed in indoor fiberglass fiber barrel (500 L) twice a day for eight weeks. The results show that the weight gain rate and the specific growth rate increased first and then decreased with increase of dietary Bio100 level, and those of T3 and T4 groups were significantly higher than those of the control group (P < 0.05). The feed coefficient rates of the experiment groups were significantly lower than that of the control group (P < 0.05). The muscle ash content of the experiment group increased with increase of dietary Bio100 level, and that of T5 group was significantly higher than those of T1 group and the control group (P < 0.05).The pepsin activity increased first and then decreased, and that of T3 group was significantly higher than those of T1, T5 groups and the control group (P < 0.05). The intestinal lipase activity increased first and then decreased, and that of T4 group was significantly higher than those of T1, T2 groups and the control group (P < 0.05). There was no significant difference in intestinal amylase activity among the groups (P>0.05). The lysozyme activity, alkaline phosphatase activity and C3 content of the experiment group were higher than those of the control group but with no significant difference (P>0.05). The Bio100 level in feed affected the cholesterol content and alanine aminotransferase activity of serum significantly. It is showed that the suitable adding amount of Bio100 in the feed of juvenile hybrid groupers was 0.15%.