We discuss an on-chip fluorescence detection system with a microchamber array based on a CMOS image sensor. The system has three main parts: the CMOS image sensor, a light guide structure to achieve high optical coupling efficiency between the fluorescence source and the photodiode in the sensor, and a microchamber array for fluorescence-tagged reaction. In this paper, we propose three types of light guide structure, a light guide array (LGA), a fiber optic plate (FOP), and a light pipe array (LPA), and compare them in terms of the fluorescent light coupling efficiency and fluorescence detection sensitivity. We found that the LPA can achieve the highest coupling efficiency and detection sensitivity. On the basis of the results, we successfully fabricated a fluorescent droplet array combined with an LPA and a polydimethylsiloxane-based microfluidic device, which is placed on a CMOS image sensor for fluorescence detection. This system can be applied to a digital enzyme-linked immunosorbent assay system that requires a microchamber array.