Peony seed oil (PSO) is an edible oil with high nutritional value. In this study, a stable oil-in-water microemulsion (Blank-ME) was prepared using PSO as the oil phase, and RH 60-CO 40-Span 80-PEG 200 (RCSP) as surfactants and co-surfactants. The physicochemical properties and stability of Blank-ME samples with different water mass fractions and RCSP/PSO ratios were studied. The average particle size and polydispersity index (PDI) decreased as the water mass fraction increased, and the PDIs of the samples containing PSO were all lower than 0.3. The visual appearance, average particle size, and PDI indicated that Blank-ME samples had good environmental stability when they were centrifuged at 10,000 r/min for 30 minutes, stored at 55 °C for 12 hours, and stored at 4 °C for 10 days. The Blank-ME could effectively encapsulate berberine hydrochloride (BH), and a BH-ME was prepared. The average particle sizes of all BH-ME samples were less than 35.00 nm, and increased as the BH concentration was increased from 0.05 to 1.0 mg/g. The system prepared with a BH concentration of 1.0 mg/g had good structural stability under different storage temperatures (4 °C, 25 °C, and 37 °C). BH and Blank-ME showed good synergy effects (32.87%) of the DPPH• scavenging capacity after 24 hours of reaction. The antitumor and antibacterial activities also exhibited possible synergism between BH and Blank-ME. These findings are positive for the application of MEs and BH in pharmaceutical, food, and other industries.