The invention relates to the field of molecular biology, and discloses an active pharmaceutical ingredient screening method based on a serotonin receptor 2A molecular probe; the method comprises steps of constructing the serotonin receptor 2A molecular probe and establishing an Flp-In T-Re* HEK293 cell line for inducing the expression of a G protein-coupled receptor molecular probe; the serotonin receptor 2A molecular probe is established by virtue of a method of fluorescent resonance energy transfer; the molecular probe has the characteristics of high sensitivity, low noise and high throughput; by virtue of the molecular probe, both receptor agonists and antagonists/inverse agonists can be screened, and the process of screening is free from interference of downstream signals; and the molecular probe, which is reasonably designed, can be used for screening traditional Chinese medicine mixtures, and the molecular probe can screen out lead compounds which are clear in molecular action targets at a receptor level by one step.