A recombinant engineered strain for de novo synthesis of CDP-choline using glucose as a substrate and its preparation method and application are provided. Using BS168N as the starting strain, firstly, the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 from S. cerevisiae are integrated into the genome of the BS168N for induced expression, thereby opening up the synthesis pathway from phosphatidylethanolamine to phosphatidylcholine; subsequently, the CKI and CCT genes of S. cerevisiae are further integrated into the BS168N genome expressing PEM1-PEM2, opening up the synthesis pathway of choline to CDPC, thereby obtaining the recombinant engineered strain. Further, the recombinant engineered strain is subjected to shake flask fermentation to achieve de novo synthesis of CDP-choline using glucose as a substrate. The method of the present disclosure provides a fundamental research and theoretical basis for the construction of efficient cell factories for de novo synthesis of CDP-choline through synthetic biology.