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Targeted tumor imaging in vivo has been one of the main challenges in the early stage of cancer diagnosis and therapy. However, few contrast probes are available to specifically respond to tumor environments. The aim was to investigate porphyrin‐1,8‐naphthalimide derivative (APTSPP‐2NI) as a potential tumor‐targeting fluorescent probe in vitro and in vivo. A tumor‐targeting APTSPP‐2NI was synthesized by the incorporation of water‐soluble 5‐(4'‐aminophenyl)‐10,15,20‐tris(4'‐sulfonato‐phenyl) porphyrin (APTSPP) as a tumor‐targeting group into N‐butyl‐4‐ethyldiamino‐1,8‐naphthalene imide (NI) fluorescence molecule herein. The derivative was further characterized and its properties in vitro and in vivo including the cell cytotoxicity, cell uptake of tumor cells and fluorescent imaging were also evaluated. Experiment results indicated that APTSPP‐2NI possessed the low cell cytotoxicity and good tumor‐targeting property to the B16F10 melanoma cells. Moreover, APTSPP‐2NI can be taken up highly by B16F10 melanoma tumors and then achieve the good green and red fluorescent imaging in B16F10 melanoma tumors. Therefore APTSPP‐2NI can be considered as a potential tumor‐targeting dual‐mode enhanced probe in fluorescent imaging in vitro and in vivo. [ABSTRACT FROM AUTHOR]