Abstract S-adenosylhomocysteine (SAH) is an effective sedative, a good sleep modulator, and a new anticonvulsant. SAH can be synthesized from adenosine and homocysteine by using microbial S-adenosylhomocysteine hydrolase (SAHase). The extremely thermostable SAHase and lactate dehydrogenase (LDH) from Thermotoga maritima were successfully overexpressed in Escherichia coli, and purified by heat treatments. The SAHase exhibited the highest activity at 85°C and pH 8.0 with a specific activity of 6.2U/mg when NAD concentration was 1mM. However, optimal SAHase reaction conditions shifted to 100°C and pH 11.2, and its specific activity increased to 36.8U/mg after NAD concentration was raised to 8mM. Biosynthesis of SAH at 85°C largely increased the adenosine solubility which was a limiting factor for improving the titer of product. At 85°C and pH 8.0, 24μmol of SAH was obtained when 0.5mg of SAHase was applied to a 10ml reaction mixture. The SAH production was further increased to 153μmol by adding LDH and pyruvate into the reaction mixture for NAD regeneration. Therefore, extremely thermostable enzymes SAHase and LDH from T. maritima form an efficient NAD consumption and regeneration system for SAH biosynthesis. This method has great potential for industrial-scale enzymatic production of SAH.