This study aimed to investigate the temperature effects on interferons (IFNs) signaling pathway and IFNs-regulated immune system in orange-spotted grouper. As climate changes resulted in temperature variation in recent years, it leads to widespread outbreaks of diseases frequently in aquaculture, which eventually cause heavy impacts on the global aquaculture industries. Fish are ectothermic animals, and hence their immune system is deeply modulated by ambient temperature changes. Therefore, understanding the molecular mechanism of how temperature changes influence the immune system in fish will contribute to developing the strategy for disease prevention and control. However, relevant research about this issue is relatively less, so this study aims to investigate the effects of temperature variation on IFNs-regulated mechanisms in orange-spotted grouper (Epinephelus coioides). In this work, with the treatment of different temperatures, the comparative analysis of type I IFNs, type II IFNs, and other related immune molecules involved in antiviral responses (Mx1, ISG15, Viperin and IFNR1) was conducted, which provided the fundamental information to explore environment temperature factors regulating antiviral molecular mechanism. The comparative analysis of the IFNs system in orange-spotted grouper along with the immune stimulation was performed at high and low temperatures respectively. The results indicated IFN-regulated genes were suppressed at the late time point upon high-temperature treatment. The cloning of Mx1, Mx2, and Mx3 promoters was done, meanwhile, we proved that temperature modulation could influence the Mx promoters’ activity and their transcript expression level. Thereby, our study provided evidence of how temperature modulation affects the IFN-regulated system for future antiviral response studies, which would be possible to develop a new strategy to overcome the impact of climate change on aquaculture.