Abstract: To deeply investigate the structural characteristics and activities of nanovesicles derived from Rosa roxburghii Tratt. (RDNs), they were isolated, extracted, and characterized. Additionally, their antioxidant, hypoglycemic, and anti-hemolytic activities against Staphylococcus aureus exotoxin were explored. The RDNs were isolated using ultracentrifugation, and their morphology was characterized through transmission electron microscopy (TEM). The protein, lipid, and RNA composition/concentrations were quantified employing the BCA assay, thin-layer chromatography (TLC), and RNA extraction kits, respectively. The antioxidant capacity was assessed using DPPH and ABTS ⁺ radical scavenging assays, while the hypoglycemic activity was evaluated through α-glucosidase and α-amylase inhibition assays. Using 2% rat erythrocytes as the research object, the experiment was divided into blank control group, positive control group, S. aureus-EVs group, RDNs group, and RDNs+S. aureus-EVs group, and in vitro anti-hemolytic rate of RDNs was detected. The results indicated that RDNs had an average hydrodynamic diameter of 155.27±1.39 nm, a zeta potential of −8.60±0.64 mV, and maintained stability at −80 ℃ for 12 days. The particles remained stable during the first 6 days of storage at 4 ℃, but exhibited significant particle size changes after surpassing the 6-day mark (P<0.01). Particle size remained unchanged (P>0.05) at pH1.2, while pH6.5 showed significant change in particle size (P<0.05). Biochemical analysis uncovered the presence of protein (5.50±0.00 mg/mL), lipid (4.23±0.40 mg/mL), and RNA (3.91±1.97 μg/mL). Notably, protein bands were observed at 24, 50~70, and 70 kDa. Lipid components exhibit an intricate composition, which includes phytosterols and lecithins. Concentration-dependent (0 to 212.30 mg/mL) scavenging of DPPH and ABTS⁺ radicals was observed, with the half-inhibitory concentrations (IC₅₀) for the two radicals being 112.90 and 64.38 mg/mL, respectively. Additionally, when the concentration reached 212.30 mg/mL, the RDNs were capable of eliminating 99.80% of ABTS⁺. The α-glucosidase and α-amylase inhibition rates were 48% and 21%, respectively, significantly lower than positive controls (P<0.01). The hemolysis rates were nearly 0 in the RDNs group, 64% in the S. aureus-EVs group, and 8% in the RDNs+S. aureus-EVs group, which was significantly lower than that in the S. aureus-EVs group (P<0.01). The RDNs had antioxidant and hypoglycemic activities and could exert anti-hemolytic effects against S. aureus exotoxins, laying a foundation for their potential applications in the fields of antioxidants and natural antibacterial agents.