Abstract: To determine the optimal dietary vitamin B₂ requirement of juvenile golden pompano (Trachinotus ovatus), six experimental diets were formulated which contained 0 mg·kg^–1, 4.4 mg·kg^–1, 6.2 mg·kg^–1, 8.0 mg·kg^–1, 11.3 mg·kg^–1 and 15.5 mg·kg^–1 of vitamin B₂. Each diet was assigned to three parallels of 25 fish initial mass of (11.63±0.17) g which were fed twice daily for eight weeks. The results show that the weight gain rate and specific growth rate of the fish fed with diet of 4.4 mg·kg^–1 vitamin B₂ were significantly higher than those of 0 mg·kg^–1 group (P<0.05), and the activities of aspartate aminotransferase and alanine aminotransferase in the serum were significantly lower than those in 0 mg·kg^–1 group (P<0.05). No significant effect was found on the fish coefficient, survival rate, liver vitamin B₂ content, serum immune parameters, crude protein and crude fat content in whole body, moisture, crude fat and crude ash in muscle (P>0.05); the liver D-AAO activity in 6.2 mg·kg^–1 group was significantly higher in 0 mg·kg^–1 group (P<0.05). Broken-line model analysis estimates that the dietary vitamin B₂ requirement of juvenile golden pompano is 4.23 mg·kg^–1 for optimal growth, and 6.24 mg·kg^–1 for maximum liver D-AAO activity.