In this study, Bacillus subtilis was used as the host to express the heterologous enzyme for biotechnological production of 2’-deoxyadenosine. The pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP) from Escherichia coli as biocatalyst to convert thymidine and adenine into 2’-deoxyadenosine with high efficiency. Firstly, the enzyme combination expression and ribosome binding site (RBS) optimization of nucleoside phosphorylases were performed, and the recombinant B. subtilis that synthesized 2’-deoxyadenosine by whole-cell catalysis was constructed. The yield of 2’-deoxyadenosine was 133.4 g/L, and the molar conversion rate of thymidine was 64.3%. Then, to further improve the yield and molar conversion rate, a short peptide pair was used to form self-assembling multienzyme complexes in vivo. The yield of 2’-deoxyadenosine was significantly improved, reaching to 179.6 g/L. Finally, the conditions of whole-cell catalysis were optimized to further improve the production of 2’-deoxyadenosine. The optimized production of 2’-deoxyadenosine reached to 200.3 g/L and the molar conversion rate of substrate thymidine was 96.6%.