D-Glucose-1-phosphate (α-G1P) is a metabolic intermediate involved in a variety of metabolic reactions, and is also a potential drug, which has great development potential in the field of medicine and health supplements. It is mainly produced by α-glucan phosphorylase (αGP) using starch as substrate. In this study, α-glucan phosphorylase ThαGP from Thermotoga sp. SG1 was heterologously expressed in Bacillus subtilis. Then the types of promoters, fermentation medium formulation, temperature and time were optimized. And the application experiment of preparing α-G1P was carried out. The results showed that the enzyme activity of recombinant strain B. subtilis WS9/pHY300PLk-SpovG-amyQ-thαgp was 7.01-fold higher than that of the original strain fermentation. After optimization of shake flask fermentation conditions, the total activity of ThαGP reached 173.38U/mL after fermentation at 33℃ for 60h in a shaking flask using medium with 18g/L tryptone and 30g/L yeast extract as complex nitrogen source and 15g/L soluble starch as carbon source, which was 4.13 times that of the original fermentation. When ThαGP was combined with pullulanase and 4-α-glucosyltransferase, the conversion rate of α-G1P catalyzed by ThαGP was up to 51.62%. It was hoped that the study would provide a reference and technical foundation for the food grade fermentation preparation of ThαGP and industrial production of α-G1P. 〖