Abstract Novel biosurfactants with high performance are always needed in the petroleum industry for environmental sustainability. Herein, we developed a series of biosurfactants to enhance the heavy oil recovery from Canadian oil sands. Pseudo-Gemini biosurfactants were designed to be interfacially active and CO2 switchable. The strong interfacial activity of biosurfactants promotes the liberation of heavy oil from solid substrates, which was demonstrated by the liberation visualization cell. On the other hand, the separation of heavy oil from extraction fluid was also facilitated by activating the CO2 switchability of biosurfactants. Since the efficiencies in both the heavy oil liberation and the oil-water separation were improved, the total heavy oil recovery could be significantly enhanced. Therefore, these biosurfactants are believed to be promising in the application of enhanced oil recovery from oil sands ore.