The monoterpenoid indole alkaloid (MIA) ajmaline, a class I antiarrhythmic drug, is derived from *Rauwolfia serpentina* (Indian Snakeroot). Despite significant progress in understanding its biosynthesis, key enzymes remained unidentified. This study identifies and characterizes two elusive reductases, RsCAD2 and RsRR4, and two root-expressing acetylajmaline esterases, AAE1 and AAE2, completing the ajmaline biosynthetic pathway. Ajmaline biosynthesis involves vomilene 1,2-reduction followed by 19,20-reduction, modulated by AAE enzymes that deacetylate 17-O-acetylornajmaline. The complete pathway is reconstituted in *Saccharomyces cerevisiae*, achieving de novo ajmaline biosynthesis. RsCAD2 and RsRR4 are rate-limiting enzymes, and increasing their expression significantly enhances ajmaline production. The study highlights the importance of these enzymes in ajmaline biosynthesis and provides a foundation for future optimization and large-scale production.The monoterpenoid indole alkaloid (MIA) ajmaline, a class I antiarrhythmic drug, is derived from *Rauwolfia serpentina* (Indian Snakeroot). Despite significant progress in understanding its biosynthesis, key enzymes remained unidentified. This study identifies and characterizes two elusive reductases, RsCAD2 and RsRR4, and two root-expressing acetylajmaline esterases, AAE1 and AAE2, completing the ajmaline biosynthetic pathway. Ajmaline biosynthesis involves vomilene 1,2-reduction followed by 19,20-reduction, modulated by AAE enzymes that deacetylate 17-O-acetylornajmaline. The complete pathway is reconstituted in *Saccharomyces cerevisiae*, achieving de novo ajmaline biosynthesis. RsCAD2 and RsRR4 are rate-limiting enzymes, and increasing their expression significantly enhances ajmaline production. The study highlights the importance of these enzymes in ajmaline biosynthesis and provides a foundation for future optimization and large-scale production.