In this study, dye/polymer matrix-stabilized β-FeOOH nanomaterials were fabricated for therapeutic applications. Rh-B/F127@β-FeOOH nanomaterials were synthesized using two different methods: co-precipitation (CoP) and hydrothermal (HT) methods. The as-synthesized nanoparticles were characterized using various spectroscopic techniques, including FT-IR, UV-Vis, PL, XRD, HR-TEM, and XPS analysis. The functional groups and optical properties were confirmed by FT-IR spectroscopy, UV-Vis and fluorescence spectroscopy. The Rh-B/F127@β-FeOOH nanomaterials exhibited both rod-like and sphere-like morphology, as confirmed by HR-TEM analysis. Unlike the nanorods, the nanospheres produced multi-colored emissions at 407, 446, 482 and 520 nm. The oxidative states and elements were confirmed by XPS spectroscopy. MTT assays were used to analyze the cytotoxicity of the nanospheres against A549 cells. The reactive oxygen species (ROS) generation and apoptotic cell death caused by the β-FeOOH nanospheres were evaluated by flow cytometry. Cell cycle analysis indicated that the treatment of nanospheres-induced S-phase cell cycle arrest in A549 cells. The synthesized nanospheres induced late-stage apoptosis in the A549 cell line, with a cell death rate of up to 30.37% at the IC