In this study, novel mesoporous siliceous vesicles with ultrathin walls (50 nm in thickness) and large cavities were synthesized through a facile biliquid-interface co-assembly method by using cetyltrimethylammonium bromide as a structure-directing agent and tetraethoxysilane as a silica source. Various synthesis parameters (stirring rate, ionic strength, reaction temperature) have been investigated and were found to affect the interface co-assembly process. The obtained vesicles are highly water-dispersible and have large mesopores (6.7 nm) in the walls, high surface area (902 m2 g-1) and large pore volume (1.57 cm3 g-1). Cytotoxicity experiments and cellular uptake studies based on fluorescence imaging indicate that the obtained vesicles possess excellent biocompatibility and can be readily internalized by Pan02 cells due to their good water dispersibility and affinity to cell membranes. The outstanding properties of the obtained mesoporous silica vesicles make them good candidates for various bio-applications, such as drug delivery and enzyme immobilization.