This study aimed to address the stability limitations of third-generation biosensors using enzymes from mesophilic organisms, by engineering a stable direct electron transfer (DET)-type dehydrogenase capable of transferring electrons extracted from the substrate to the electrode. A fusion protein combining the mediated electron transfer (MET)-type aldose sugar dehydrogenase from the hyperthermophile Pyrobaculum aerophilum (PaeASD), which cannot transfer electrons generated by enzymatic reactions to the electrode without a mediator, and the natural electron transfer protein cytochrome b562 (cyt b562) was developed to investigate its potential for the DET reaction. A recombinant protein expression system was established in Escherichia coli to produce the PaeASD-cyt b562 fusion protein, which was purified from the soluble fraction of the host cells. Intramolecular electron transfer from pyrroloquinoline quinone (PQQ) to the heme group within the PaeASD-cyt b562 fusion protein was investigated using UV-Vis absorption spectroscopy. Upon the addition of glucose, an increase in absorption corresponding to reduced heme molecules was observed, indicating electron transfer from glucose to the heme group in the cyt b562 component via PQQ in the PaeASD component. The DET capability of the fusion protein was evaluated using cyclic voltammetry with screen-printed carbon electrodes. A glucose concentration-dependent increase in current response confirmed DET activity. Notably, the fusion protein retained over 80% of its initial current response even after 2 months of storage at 4 °C. The novel robust PaeASD-cyt b562 fusion protein demonstrated efficient DET capability, highlighting its high potential for application in the development of third-generation biosensors.