Functional particles self-assembled from polymer single chain nanoparticles have potential applications in various fields, but functionality incorporation is difficult due to their ultrasmall sizes. Here, the preparation and self-assembly of amphiphilic polymer single chain Janus nanoparticles (SCJNPs) with tunable liquid crystalline (LC) properties are reported via intrachain photo-cross-linking stilbene-containing amphiphilic block copolymers (BCPs). The cross-linking degree can be controlled by varying UV irradiation time, which affects the LC properties and the size of the resulting SCJNPs accordingly. The self-assembly behavior of the LC-SCJNPs in solution was further investigated systematically. The morphology transition from nonspherical assemblies, including tubular, doughnut-like, and saddle-shaped, to spherical nanoparticles was observed by changing UV irradiation time and the composition of BCPs. Furthermore, the assemblies display a smectic phase with clear ordering stripes under TEM observation. The LC properties gradually diminish with the increasing UV irradiation time. The internal LC ordering has an important effect on the morphology transition from nonspheres to spheres. This work provides a facile approach to polymer particles with tunable functionalities that can be used for advanced materials by remote light manipulation.