Enzymes fixed on the electrode of biosensor are gradually inactivated and the electrode is discarded after using several times. In order to prepare the stable biosensor, we try to use a stable enzyme from extreme thermophilic bacteria, Thermus thermophilus HB8. It is very important that a stable enzyme from T. thermophilus HB8 is overproduced in Escherichia coli, for the purpose of enough supply of enzyme. Thereby, we determined the important sequence for overexpression of NADH oxidase (nox) gene from T. thermophilus HB8 in E. coli. As a result, it is revealed that ten nucleotides sequence, GAAATTAACT, in the upstream of start codon of nox gene was important for its overexpression in E. coli.