Two‐dimensional organic semiconductor single crystals (2D OSSCs) represent the promising candidates for the construction of high‐performance electronic and optoelectronic devices due to their ultrathin thicknesses, free of grain boundaries, and long‐range ordered molecular structures. In recent years, substantial efforts have been devoted to the fabrication of the large‐sized and layer‐controlled 2D OSSCs at the liquid‐liquid interface. This unique interface could act as the molecular flat and defect‐free substrate for regulating the nucleation and growth processes and enabling the formation of large‐sized ultrathin 2D OSSCs. Therefore, this review focuses on the liquid–liquid interface‐assisted growth methods for the controllable preparation of 2D OSSCs, with a particular emphasis on the advantages and limitations of the corresponding methods. Furthermore, the typical methods employed to control the crystal sizes, morphologies, structures, and orientations of 2D OSSCs at the liquid–liquid interface are discussed in detail. Then, the recent progresses on the 2D OSSCs‐based optoelectronic devices, such as organic field‐effect transistors, ambipolar transistors, and phototransistors are highlighted. Finally, the key challenges and further outlook are proposed in order to promote the future development of the 2D OSSCs in the field of the next‐generation organic optoelectronic devices.