The present study investigated the effect of stocking density and dietary carbon sources on the water quality, oxidative status, and immune-related genes of Nile tilapia (Oreochromis niloticus) reared under biofloc conditions (BFT). Eight groups were established at two levels of stocking densities (140 fish per m³: low stocking density, LSD) and (280 fish per m³: high stocking density, HSD) (5.15 ± 1.12 g) and kept in eight biofloc units containing water without carbon sources (control groups) or with glycerol, molasses, or starch. Red blood cells count, hemoglobin, and hematocrit values were reduced in fish stocked in control groups at LSD and HSD than biofloc groups. Control fish groups reared at both LSD and HSD have the highest significant (p < 0.05) white blood cells number than other fish groups. Meanwhile, fish groups that received glycerol, molasses, and starch maintained in both LSD and HSD presented a higher significant (p < 0.05) monocyte % than in the control group reared at both LSD and HSD. The fish group reared in biofloc conditions (BFT) using starch carbon source and reared at the HSD presented a significantly higher (p < 0.05) increase in total serum protein and albumin levels as well as globulin value than the control fish group reared at both LSD and HSD. The highest glucose and cortisol levels were showed in the control fish group reared at both LSD and HSD. Fish maintained in glycerol-based biofloc at LSD attained the highest (p < 0.05) serum superoxide dismutase (SOD), glutathione reductase (GR), and catalase than other experimental groups. Regarding the nonspecific immune status, significantly increased expression of CC-chemokines, CXC-chemokines, TLR7 and IL-8 genes was found in molasses based biofloc groups. The data of the present study revealed that using molasses promotes health status of Nile tilapia cultured in a biofloc system.