In order to explore the effects of dietary calcein (CAL) for fluorescence labeling on the levels of antioxidant enzymes, and lipid peroxidation in different tissues, juvenile Spinibarbus sinensis were fed continuously with different CAL dosages feed (including 0, 2, 8, and 32 g/kg) for 16 days (i.e., toxicity accumulation experiment), and then with feed without CAL for 32 days (ie., toxicity elimination experiment). Moreover, the recovery after CAL feeding, as well as the toxic effects of CAL were further evaluated. The results of toxicity accumulation experiment showed that the excess reactive oxygen species (ROS) could be successfully removed by the antioxidant system of juvenile S. sinensis, when the CAL dosage ≤ 32 g/kg and the cumulative feeding duration ≤ 8 days. However, long feeding period (e.g., 16 days) might cause toxic effects, and even lead to excessive oxidative stress in hepatopancreas and kidney of experimental fish. The results of toxicity elimination experiment showed that most of the antioxidant enzyme indexes, non-enzyme antioxidants and lipid peroxidation products in serum, hepatopancreas and kidney tissues of juvenile S. sinensis recovered to safe levels after 32 days. The present study confirmed that the toxic effects of CAL feeding in juvenile S. sinensis depended on cumulative feeding duration when dosage ≤ 32 g/kg. In other words, when CAL is used to mark juvenile S. sinensis by feeding, the CAL dosage and the feeding duration should be restricted to ≤ 32 g/kg and ≤ 8 days, respectively, and the marked juveniles should be reared for at least 32 days. The results are of important guiding significance for the safe and effective application of CAL in fish fluorescent labeling.