Real-time monitoring of gene expression by a bioluminescence reporter gene is a powerful method for large-scale, detailed analysis of gene expression in living cells and large-scale screening of mutants. We have developed a portable, compact, integrated automatic bioluminescence-monitoring apparatus that can continuously monitor 960 individual plant seedlings or micro-organism colonies under uniform light conditions at temperatures up to 50 °C. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms of Arabidopsis reporter strain. Using the apparatus, we measured bioluminescence rhythms in the thermophilic cyanobacterium Thermosynechococcus at temperature up to 43 °C. We also monitored the expression of the flowering regulator gene CONSTANS in Arabidopsis as bioluminescence in high time resolution under different photoperiodic conditions. The high-throughput bioluminescence-monitoring apparatus developed here is a powerful tool for real-time monitoring of gene expression and gene function.