The serotonin receptor 5-HT is widely expressed in the central nervous system and has been considered a drug target in a variety of cognitive and psychiatric disorders. The anti-inflammatory effects of 5-HT agonists may present a promising approach for Alzheimer's disease (AD) treatment. Herbal antidepressants used in the treatment of AD have shown functional overlap between the active compounds and 5-HT receptor stimulation. Therefore, compounds in these medicinal plants that target and stimulate 5-HT deserve careful study. Molecular docking, drug affinity responsive target stability, cellular thermal shift assay, fluorescence resonance energy transfer (FRET), and extracellular regulated protein kinases (ERK) 1/2 phosphorylation tests were used to identify emodin-8-O-β-d-glucopyranoside (EG), a compound from Chinese medicinal plants with cognitive deficit attenuating and antidepressant effects, as an agonist of 5-HT. EG selectively targeted 5-HT and activated the 5-HT-induced signaling pathway. The activated 5-HT pathway suppressed tumor necrosis factor (TNF)-α levels, thereby protecting neural cells against beta-amyloid (Aβ)-induced death. Moreover, the agonist activity of EG towards 5-HT receptor, in FRET and ERK1/2 phosphorylation, was antagonized by SB 224289, a 5-HT antagonist. In addition, EG relieved AD symptoms in transgenic worm models. These results suggested that 5-HT receptor activation by EG positively affected Aβ-related inflammatory process regulation and neural death resistance, which were reversed by antagonist SB 224289. The active compounds such as EG might act as potential therapeutic agents through targeting and stimulating 5-HT receptor for AD and other serotonin-related disorders. This study describes methods for identification of 5-HT agonists from herbal compounds and for evaluating agonists with biological functions, providing preliminary information on medicinal herbal pharmacology.