Paeonol, a monoterpene glycoside compound, has extensive pharmacological activities. However, its applications are restricted by poor water solubility and low bioavailability. In this study, paeonol ethosomes (PAE-ethosomes) were successfully prepared with a microfluidic method by optimizing the single factors and RSM test. The enhanced PAE-ethosomes were assessed using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), vesicle size (VS), zeta potential (ZP), and polydispersity index (PDI). Density functional theory analysis was employed to verify the molecular interaction. The optimized RSM conditions were a phospholipid concentration of 6 mg/mL, a cholesterol concentration of 1 mg/mL, and a total flow rate of 600 μL/min with a presumed value of 60.3% and confirmation results of 61.2 ± 0.3%. The prepared PAE-ethosomes showed better storage stability and a slow-release effect. The of PAE-ethosomes rose from 167.0 ± 15.8 to 272.0 ± 16.4 μg/cm after 24 h, which was substantially greater than that from a 25% hydroethanolic solution of paeonol, according to in vitro skin retention and transdermal absorption. The of PAE-ethosomes in the skin increased by 225% with 265.5 ± 15.4 vs 81.8 ± 8.2 μg/cm, compared with 25% hydroethanolic solution of paeonol. Molecular interaction between paeonol and lecithin by Gaussians showed that the paeonol compound may have a higher probability of spreading in the hydrophilic phosphate group ("head") position for the PAE-ethosomes. The () transgenic zebrafish results showed that PAE-ethosomes had better anti-inflammatory effects than paeonol. The microfluidic approach was efficient with good characteristics in physics and pharmacology with the potential in pharmaceutical use.