Betulin is a kind of pentacyclic triterpenoid extracted from the bark of Betula platyphylla, which has the characteristics of anti-inflammatory, antibacterial, antioxidant and so on. However, its poor water solubility limits its application. Betulin/MβCD inclusion complex nanofibers (Betulin/MβCD-IC-NF) were prepared by Methyl-beta cyclodextrin (MβCD) inclusion of betulin and electrospinning techniques. Scanning electron microscopy (SEM) showed that the independent Betulin/MβCD-IC-NF surface was bead-free and smooth. The findings from X-ray diffraction, nuclear magnetic resonance hydrogen spectroscopy, and Fourier transform infrared spectroscopy displayed that an inclusion complex was synthesized containing betulin and MβCD. The results of thermogravimetric analysis displayed that Betulin/MβCD-IC-NF has better stability. The phase solubility study proved that MβCD had the effect of increasing the solubility of betulin. In addition, the DPPH free radical scavenging test and the antibacterial test showed good antioxidant properties and antibacterial properties of Betulin/MβCD-IC-NF, respectively. In conclusion, Betulin/MβCD-IC-NF with high water solubility was obtained by electrospinning, and its thermal stability, antibacterial and antioxidant activity were improved.