Two-photon photodynamic therapy (TP-PDT) offers a targeted, minimally invasive approach for cancer treatment by generating reactive oxygen species (ROS) that selectively eliminate tumor cells. In this study, we present a heavy atom-free, azo-based photosensitizer (PS) that is co-assembled with the RGD (Arg-Gly-Asp) peptide to facilitate αvβ₃ integrin-targeted delivery. The PS, synthesized through a straightforward azo coupling approach, self-assembles into nanoparticles (NPs) exhibiting excellent colloidal stability, well-defined emission characteristics, and enhanced type-I ROS generation efficiency. The resulting NPs show preferential cellular uptake and robust two-photon-induced phototoxicity against colorectal cancer cells, while minimizing effects on normal cells. Notably, the application of TP-PDT to human colorectal biopsy samples demonstrated that these NPs can differentiate malignant from healthy tissue and achieve selective tumor ablation. Collectively, these findings highlight the potential of integrating TP-PDT with azo-dye assemblies as an advanced theragnostic strategy for early-stage detection and treatment of colorectal cancer.