Abstract: Salidroside (SAL) is a phenolic substance with high solubility and low permeability, which make it easy to cause the efflux effect of P-glycoprotein and degradation of intestinal flora, resulting in lower bioavailability. The aim of this study was to develop and optimize a water-in-oil nanoemulsion of SAL (w/o SAL-N) to explore its suitability in oral drug delivery systems. In this work, SAL-N was successfully prepared by water titration method at K_m = 1 to construct the pseudo-ternary phase diagrams. Physical characterization including the average viscosity, pH, refractive index, particle size, PDI, TEM, DSC, the content of SAL, and stability study were performed. It was evaluated for drug release in vitro and pharmacokinetic studies in vivo. The optimized nanoemulsion formulation consisted of Labrafil M 1944CS (63%), Span-80/Tween-80/EtOH (27%) and 200 mg∙mL⁻¹ SAL solution (SAL-SOL) (10%). Low viscosity and suitable pH were expected for the nanoemulsion. The spherical morphology and nanoscale size of SAL-N enhanced the stability of the nanoemulsion system. In vitro drug release showed that SAL-N had a better controlled release property than SAL-SOL at earlier time points. The pharmacokinetic studies exhibited that SAL-N had significantly higher in t_1/2 (2.11-fold), AUC_{0−48 h} (1.75-fold) and MRT_{0−48 h} (2.63-fold) than SAL-SOL (P < 0.01). The w/o SAL-N prepared in this work can be effectively delivered via the oral route. It can be seen w/o nanoemulsion is a strategy for the drug with polyphenols to delay the release, enhance oral absorption and reduce metabolic rate.