Biofloc technology (BFT) is an environmentally friendly aquaculture technology and has been demonstrated to improve water quality and promote the growth, immunity, disease resistance, and reproduction of some cultured animals. The lined seahorse (Hippocampus erectus) has been cultured in China for more than 10 years; however, there are still some major problems in large-scale culture, one of which is that juveniles are prone to enteritis. In this study, to test the feasibility of BFT for H. erectus juvenile, the effects of different carbon sources (glucose, sucrose, or corn starch) and carbon-nitrogen ratios (C/N, 16:1 or 20:1) on the water quality, survival, growth, and intestinal biochemical parameters of juveniles cultured under zero-water exchange conditions were investigated. The results show that different combinations of carbon source and C/N had different effects. Some combinations had significant positive effects, while others had no or even negative effects. The best positive effects appeared in combinations of glucose and C/N 16:1, and sucrose and C/N 16:1, which were mainly reflected in the reduction of ammonia and nitrite in water, the promotion of bacterial proliferation and community diversity in water; and the improvement in intestinal microflora, digestion, immunity and survival of the juveniles. In conclusion, adding extra organic carbons to water is promising to improve the culture of H. erectus juveniles, as long as the carbon source type and C/N are properly combined (e.g., glucose or sucrose at C/N 16:1). This study may provide a new potential method for seahorse enteritis.