At present, marine aquaculture in China and other countries around the world is mainly carried out in shallow coastal waters, which has caused a series of environmental problems and affected the sustainable development of aquaculture. Chinese researchers have been working on salmon mariculture far offshore in the Yellow Sea, and rainbow trout (Oncorhynchus mykiss) is one of the popular salmonid. Light color, which can affect the entire life cycle of aquatic species, is an important factor in the management of far offshore cultures. The sunlight irradiated on the water surface enters the water body after refraction, and the incident light will be absorbed and scattered strongly by the water and will attenuate rapidly. Blue light has strong penetrating power in clean sea water. Red light and ultraviolet light attenuate rapidly when passing through clean water. It is important to explore the effects of light color on the antioxidant enzyme activities and related hormone concentrations of O. mykiss, which will provide important information for the practical management of far offshore cultures. O. mykiss were injected with different concentrations of melatonin (0 and 3 mg/mL) and were exposed under four light colors (white, red, green, and blue) for 28 hours. Totally eight treatments were set up i. e. white light group of control, red light group of control, green light group of control, blue light group of control, white light group treated with melatonin, red light group treated with melatonin, green light group treated with melatonin, and blue light group treated with melatonin. O. mykiss (42.0±1.5) g were cultured in twenty-four tanks (PVC, length×height×width: 80 cm×60 cm×50 cm) and all tank sides were covered with opaque covers, light sources were placed above the surface of water. Serum and head kidney tissues of O. mykiss were collected at 4, 8, 12, 16, 20, 24, and 28 hours of the experiment to detect superoxide dismutase (SOD), cortisol, and melatonin in serum and catalase (CAT) in head kidney. The results showed that the activities of SOD and CAT, and the concentrations of cortisol were higher at 16 hours and were stable from 24 to 28 hours during the study. The average activities of SOD, CAT, and the average concentrations of cortisol in red light groups were significantly higher than the other light groups (P < 0.05). And the average activities of SOD and the average concentrations of cortisol in green light groups were significantly lower than the other light groups. The average activities of CAT in green light groups were significantly lower than the red and blue light groups. The SOD and CAT activities and cortisol concentrations in O. mykiss injected with melatonin showed a down trend during the experiment. Compared with the non-injection melatonin groups, melatonin injection made the activities of SOD and CAT and the concentrations of cortisol decrease significantly and the concentrations of melatonin increase significantly in O. mykiss. This study indicated that in a 28-hour experiment, O. mykiss were strongly stressed by light at 16 hours, and then the fish adapted to the light environment; red light stressed O. mykiss and green light did not produce stress; injection melatonin could release red light pressure for fish. In sum, green light and intraperitoneal melatonin injection could be used in O. mykiss production to relieve the fish oxidative stress response. Red light stresses fish and is unsuitable for fish cultivation. Long-term experiments should be carried out to study the effects of different light colors on the growth, physiological, and biochemical responses of O. mykiss.