Albiflorin, a traditional Chinese herb, is a main component of Radix paeoniae Alba, which has been used for the treatment of depressive disorders since ancient times. However, the mechanism of the antidepressant effect of albiflorin is poorly understood. Thus, we explored the binding profile of albiflorin at neurotransmitter receptors and transporters. We also characterised the in vivo effect of albiflorin on monoaminergic systems by using microanalysis to determine the extracellular levels of serotonin (5-HT) and norepinephrine (NE) in the hypothalamus of freely moving rats administered albiflorin. We found that albiflorin inhibited the uptake of 5-HT and NE and displayed robust binding affinities for the transporters of both neurotransmitters. By contrast, albiflorin (10 μM) showed no significant affinity to a wide array of central nervous system receptors. The results of our in vivo microdialysis studies showed that administration of albiflorin (3.5, 7.0, 14.0 mg/kg) significantly increased extracellular concentrations of 5-HT and NE in the hypothalamus of freely moving rats. Overall, the current study showed that albiflorin is a novel 5-HT and NE reuptake inhibitor with high selectivity.