Abstract: Rhamnolipid is widely used as biosurfactant due to its good surface activities and environment-friendly characteristics. However, its application in daily chemical industry is limited by their poor color and difficulty in storage and transportation. In this paper, rhamnolipid calcium with light color was prepared from rhamnolipid, and its structure, surface activity and physicochemical properties were studied. ¹H NMR and FT-IR analysis revealed that rhamnolipid calcium had the same main structure as rhamnolipid. The content analysis showed that it was composed of 3.12% (mass fraction) calcium ions and 36.22% (mass fraction) rhamnose, and the molar ratio of calcium to rhamnolipid was 1:2. Thermogravimetric analysis exhibited that the melting point of rhamnolipid calcium was about 125 ℃, which was much higher than that of rhamnolipid (83 ℃), while their thermal decomposition stability was similar. In addition, rhamnolipid calcium could reduce the surface tension of aqueous solution to about 27 mN⋅m⁻¹ at concentrations above the critical micelle concentration (CMC) of 223.7 mg⋅L⁻¹, which had a surface activity similar to rhamnolipid. Hence, rhamnolipid calcium should be an ideal biosurfactant suitable for daily chemical industry.