Particle self-assembly enables construction of ordered architectures with properties distinct from their individual building blocks. Incorporating particles with diverse functionalities can generate novel collective behaviors, while multi-level assemblies bridge microscopic order to macroscopic structures. Among anisotropic building units, Janus particles are widely studied, yet conventional rigid, large-sized spheres often yield irregular aggregates due to dominant isotropic van der Waals forces and limited contact areas. We demonstrate that small, flexible polymeric Janus particles overcome these drawbacks by enhancing anisotropic interactions, promoting fusion at attractive regions, and stabilizing complex architectures. Our research achieves efficient synthesis of highly regular polymeric Janus nanoparticles and their controlled assembly into ordered multi-level structures. Furthermore, we develop single-chain Janus particles, integrating polymer chain flexibility with nanoscale anisotropy, enabling tunable composition and geometry. These advances provide new strategies for precise hierarchical organization and functional material design.