In Taiwan, the fourfinger threadfin (Eleutheronema tetradactylum), which is not resistant to low temperatures and is susceptible to dying from cold damage, is an important high-value farmed fish. Therefore, the purpose of this study was to identify nutritional supplements that could improve the cold tolerance of the fourfinger threadfin. Five nutrient supplements, including vitamin E, choline, vitamin C, tryptophan, and vitamin C+E complex, were chosen and fed to the fourfinger threadfin for four weeks before the fish were subjected to cold stress (25 minutes at 14°C). Compared to the control group, the results showed that the cold tolerance of fish increased by 18, 55, 64, 68, and 59%, respectively (p < 0.01). On the basis of the above results, three single nutrients, namely choline, vitamin C, and tryptophan, were selected and fed to fish for 1, 2, and 3 weeks, respectively, before they were challenged with cold stress. The results showed that the tolerance of the fish to cold stress did not differ between all groups after feeding for 1 week; the choline and vitamin C supplemented groups were significantly better than the control group (p < 0.05), but had no difference with the tryptophan supplemented group when fish was fed for 2 weeks; all the supplemented groups were significantly better than the control group (p < 0.01), and as compared to the control group, In addition, the five nutritional supplements listed above can significantly increase fish weight gain (p < 0.01). Choline, vitamin C, and tryptophan can effectively reduce fish blood cortisol levels.