On-site detection by flow-through polymerase chain reaction (PCR) microfluidic systems for rapid and highly sensitive analysis, are significantly desired for bioanalytical and medical research. The conventional continuous-flow PCR chips realized rapid detection, but their sensitivity was very low (10(6) to 10(8) copies μL(-1)). We improved this drawback by coating the chip with a PCR reagents mixture, and succeed to obtain a rapid and highly sensitive detection by using a segment-flow PCR system. In the present work, we developed a portable segment-flow PCR system for practical use. PCR was performed for the uid A gene in E. coli. By real-time segment-flow PCR using coated chips, we realized rapid detection in 8 min and a high sensitivity of 4 cells μL(-1). The sensitivity by the segment-flow PCR chip was the same as that of a conventional thermal cycler. Moreover, the detection speed of our segment-flow PCR chip was 15-times as rapid as that of the conventional thermal cycler.