In this study, two water-soluble red fluorescent protein (RFP) dimers, FP 2 R' and FP 2 R'', were synthesized by linking two phenothiazine-based RFP chromophore analogues through alkyl chains or alkoxy chains for hypoxic two-photon photodynamic therapy. RFP dimers are heavy-atom-free two-photon photosensitizers in which the intersystem crossing process is boosted by S and N heteroatoms. In terms of the aqueous solubility, the saturation concentration of FP 2 R'' was 3.5 mM, the emission wavelength was 677 nm, the singlet oxygen yield was 18%, and the two-photon absorption coefficient ( β ) was 2.1 × 10 -11 cm W -1 . Further, the RFP dimer FP 2 R'' showed excellent biocompatibility, negligible dark toxicity, and could produce 1 O 2 and O 2 ˙ - simultaneously. Under 460 nm illumination, the photosensitizer FP 2 R'' showed high phototoxicity with an IC 50 value of 4.08 μM in an hypoxia environment, indicating that the photosensitizer FP 2 R'' has an excellent anti-hypoxia ability. In addition, the photosensitizer FP 2 R'' demonstrated a precise localization ability to lysosomes and its Pearson's colocalization coefficient was 0.94, which could guide the aggregation of photosensitizers in the lysosomes of tumor cells to effectively improve its photodynamic therapy (PDT) effect. In particular, when exposed to 800 nm two-photon excitation, FP 2 R'' effectively produced 1 O 2 and O 2 ˙ - in zebrafish and exhibited a bright two-photon fluorescence imaging capability. At the same time, the efficacy of two-photon photodynamic therapy mediated by the photosensitizer FP 2 R'' was verified in the tumor zebrafish model, and the growth of tumor cells in zebrafish was significantly inhibited under a two-photon laser irradiation. The water-soluble two-photon photosensitizer FP 2 R'' that was reasonably constructed in this study can be used as a high-efficiency hypoxic two-photon photosensitizer to inhibit deep tumor tissues.