Background: Ginsenoside Rg5 has been proven to possess numerous health benefits. However, Rg5 is difficult to prepare using the current methods, and the poor stability and solubility of Rg5 are intractable properties that limit its application. We try to establish and optimize a new method for preparing Rg5. Methods: Different amino acids acted as catalysts, and reaction conditions were investigated to transform Rg5 in GSLS. Different CDs and reaction conditions were investigated for the preparation of CD-Rg5 based on yield and purity; ESI-MS, FT-IR, XRD and SEM analyses were used to prove the formation of the CD-Rg5 inclusion complex. Both the stability and bioactivity of β-CD-Rg5 were investigated. Results: The content of Rg5 reached 140.8 mg/g after transformation of GSLS using Asp as a catalyst. The yield of β-CD-Rg5 reached a maximum of 12% and a purity of 92.5%. The results showed that the β-CD-Rg5 inclusion complex can improve its stability of Rg5 against light and temperature. Antioxidant activity analyses against DPPH, ABTS+, and Fe2+chelation showed enhanced antioxidant activity of the β-CD-Rg5 inclusion complex. Conclusions: A novel and effective strategy for the separation of Rg5 from ginseng stem–leaf saponins (GSLS) was developed to improve the stability, solubility, and bioactivity of Rg5.