Molecular assembly is crucial in functional molecular materials and devices. Among the molecular interactions that can form assemblies, stacking among π-conjugated molecular backbones plays an essential role in charge transport through organic materials and devices. The single-molecule junction technique allows for the application of an electric field of approximately 108 V/m to the nanoscale junctions and to investigate the electric field-induced assembly at the single-stacking level. Here, we demonstrate an electric field-induced stacking effect between two molecules using the scanning tunneling microscope break junction (STM-BJ) technique and we found an increase in the stacking probability with increasing intensity of the electric field. The combined density functional theory (DFT) calculations suggest that the molecules become more planar under the electric field, leading to the energetically preferred stacking configuration. Our study provides a new strategy for tuning molecular assembly by employing a strong electric field.