At present, ulcerative colitis (UC) has become a global disease due to its high incidence. Hyperoside (HYP) is a naturally occurring flavonoid compound with many pharmacological effects. This study aimed to develop HYP-loaded mixed micelles (HYP-M) to improve oral bioavailability of HYP and to evaluate its therapeutic effect on UC. The prepared HYP-M exhibited stable physical and chemical properties, smaller particle size (PS) (21.48 ± 1.37 nm), good polydispersity index (PDI = 0.178 ± 0.013), negative Zeta potential (ZP) (- 20.00 ± 0.48 mV) and high entrapment rate (EE) (89.59 ± 2.03%). In vitro release and in vivo pharmacokinetic results showed that HYP-M significantly increased the releasing rate of HYP, wherein its oral bioavailability was 4.15 times higher than that of free HYP. In addition, HYP-M was more effective in the treatment of UC than free HYP. In conclusion, HYP-M could serve as a novel approach to improve bioavailability and increase anti-UC activity of HYP.