Precise and independent control of illumination and temperature is essential for photobiological experiments and mammalian cell culture. To overcome the limited throughput and thermal instability of existing lighting incubators, we developed a high-throughput lighting incubator comprising eight independently controlled light-exposure chambers within a shared physiological environment. The integration of high-density LED arrays in such a confined architecture, however, leads to severe heat accumulation, making it difficult to maintain the required 37 °C operating condition. Here, we report the design, optimization, and experimental validation of an active liquid-cooling thermal management system tailored for this multi-chamber instrument platform. Guided by three-dimensional computational fluid dynamics simulations, a serpentine liquid cooling plate was optimized and implemented to replace conventional passive fin heat sinks, which were found to cause substrate temperatures exceeding 45 °C under high-power operation. The assembled instrument, coupled with an industrial chiller for precise coolant temperature control, was systematically characterized. Experimental results demonstrate that the chamber temperature can be stably maintained at 37 ± 0.5 °C under continuous high-power illumination, with minimal inter-chamber variation over long-term operation. This instrument provides a robust and reproducible platform for high-throughput photobiological experiments requiring strict thermal stability and independent multi-parameter optical control.