Fluorescent nanoparticles (NPs) with aggregation-induced emission (AIE) characteristics hold remarkable potential for image-guided phototherapy. The molecular packing is the key point for optimizing the performance of AIE luminogens (AIEgens) in the aggregated or solid state. However, so far, the packing mode of AIEgens in NPs is still vague, causing some challenges for understanding the relationship between the photophysical property and packing mode, as well as further optimizing the performance of NPs for biomedical applications. In this contribution, by simply controlling the length of alkoxy chains in the donor–acceptor conjugated OPTPA, a packing balance between the twisted molecular structure and effective π-conjugation is actualized. Subsequently, the possibility of amorphous-crystalline transition of AIEgens in the polymer-encapsulated NPs is presented for the first time, and the comprehensive performance of NPs is further optimized. Both in vitro and in vivo experiments indicate that crystalline AIEgen-based NPs are remarkably effective in trimodal imaging-guided synergistic phototherapy.