We developed a real-time optical monitoring system consisting of a monochrome complementary metal-oxide semiconductor (CMOS) camera and two light-emitting diodes (LEDs) with a constant temperature incubator for the rapid detection of microbial growth on solid media. As a target organism, we used Alicyclobacillus acidocaldarius, which is an acidophilic thermophilic endospore-forming bacterium able to survive in pasteurization processes and grow in acidic drink products such as apple juice. This bacterium was cultured on agar medium with a redox dye applied to improve detection sensitivity. On the basis of spectroscopic properties of the colony, medium, and LEDs, an optimal combination of two LED illuminations was selected to maximize the contrast between the colony and medium areas. We measured A. acidocaldarius and Escherichia coli at two different dilution levels using these two LEDs. From the results of time-course changes in the number of detected pixels in the detection images, a similar growth rate was estimated amongst the same species of microbes, regardless of the dilution level. This system has the ability to detect a colony of approximately 26 μm in diameter in a detection image, and it can be interpreted that the size corresponds to less than 20 μm diameter in visual inspection.