Temperature is known to affect both the structural and functional properties of proteins in ectothermic animals like fish. Habitat temperature can lead to oxidative stress and influence the metabolic rates of enzymes in fish. In the current study, juvenile Chinese sturgeon (Acipenser sinensis), an anadromous and threatened species that lives only in the Yangtze River, were cultured under laboratory conditions for 66 days at 15, 20, 25, and 30 °C. We then studied the effects of temperature on the oxidative stress biomarkers in juvenile Chinese sturgeon. We found the activity of lysozyme (LSZ) reached its maximum at 25 °C (30.1 ± 1.2 μg/mL), while it reached its minimum at 15 °C (13.1 ± 3.3 μg/mL). In addition, the activity of xanthine oxidase (XOD) reached its maximum at 30 °C (15.20 ± 3.50), while it reached its minimum at 25 °C (12.01 ± 1.66 U/L). Furthermore, both the ability of inhibiting hydroxyl radicals (AIHR) and total antioxidative capacity (T-AOC) were increased at first and subsequently decreased with increasing temperatures, and both reached their maximum at 20 °C (1344.9 ± 349.2 U/mL and 9.54 ± 0.36 U/mL, respectively). Both AIHR and T-AOC were significantly higher at 20 °C than their corresponding levels at 25 °C and 30 °C. These results indicate that the temperature stress was higher at 15 °C and 30 °C for juvenile Chinese sturgeon. Based on the exhibited levels of LSZ, XOD, AIHR, and T-AOC in fish, we conclude the temperature range of 20−25 °C caused the least stress on the fish, and should be considered as the appropriate growth temperature for juvenile Chinese sturgeon.