Background: Nano-Se-enriched Gracilaria lemaneiformis (G. lemaneiformis) as feed ingredient on aquatic animals remained largely unknown. Feeding experiment was conducted for a total of 45 days to research the biological impacts of nano-selenium (Nano-Se) (group N), G. lemaneiformis (group G) and Nano-Se-enriched G. lemaneiformis (group NG) on growth, selenium concentration, oxidation, immunity and nutrition of Epinephelus coioides.Results: At each tested time point, the weight gain rate, specific growth rate and feed conversion rate of group N, group G and group NG were significantly increased in contrast to those of the control group (group C). A remarkable increase of the hepatosomatic index (HSI) and viscerosomatic index (VSI) was observed in group N and group NG. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of fish in group N, group G and group NG were significantly enhanced relative to those in group C. The immune system was also activated by showing an elevated expression level of various immunological genes including Immunoglobulin M, tumor necrosis factor-α and Interleukin -8. The muscle Se content in group N and group NG was 31- and 40.5-fold that of the control group (group C), respectively. The liver Se value of group N and group NG was 1.06 and 2.50 times higher than that of group C, respectively. The lipid and moisture values of group N and group NG are increased and reduced, respectively, in comparison to that of group C. The diet supplementation of Nano-Se-enriched G. lemaneiformis led to significant increase of the protein content, delicious amino acids along with total amino acids content in grouper. Conclusions: Our results demonstrate a great application potential of Nano-Se-enriched G. lemaneiformis as dietary additive in grouper.