Based on the transfer vector pPICZαA the recombinant plasmid DNA pPICZαA-TP (4862 bp) containing cDNA (deoA gene, 1326 bp) of the Thymidine phosphorylase (TP) of E. coli were constructed. The cloned plasmid pPICZαA-TP used for expression of recombinant thymidine phosphorylase in Pichia pastoris yeast. The substrate specificity of the corresponding recombinant enzyme was studied by hydrolysis of thymidine (2-Deoxy-β-D-ribofuranosyl) thymine) to thymine and 2-deoxy-β-D-ribofuranosyl. The recombinant thymidine phosphorylase (≈49 kD) expressed in Pichia pastoris GS115 cells exhibits high hydrolytic activity and can be used for enzymatic transglycosylation of nucleosides.