A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 μm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.