The carbon and artificial substrate integration was applied in mudskipper (Pseudapocryptes elongatus) culture to evaluate its effect on growth and enzyme activity. Wild mudskipper fries (6.9–7.3 cm; 3.88–4.12 g) were obtained in the coastal area of the Mekong delta. Fish were reared in 1 m3 fiber glass tanks containing 600 L of 15‰ seawater for 60 days in outdoor conditions. Mud was added to the tank bottom around 15 cm to simulate the culture condition as would be found in an earthen pond. Molasses (38% Carbon) was added daily to the culture water based on TAN level to balance C/N to 10. The results showed that the survival was in the range of 80.6–85%, and no significance in term of survival was found when comparing treatments. The individual weight of fish was 11.93–14.11 g/ind in range, fish in the culture where carbon and both carbon and substrate addition had significantly better growth and productivity than fish in the culture where molasses and substrates were not applied. The results of this study demonstrate that the growth performance and production of mudskipper significantly increased when the biofloc and substrate integration was applied.