The development of novel photosensitizer with high phototoxicity, low dark toxicity, and good water solubility is a challenging task for photodynamic therapy (PDT). A series of chlorin p6-based water-soluble amino acid conjugates were synthesized and investigated for antitumor activity. Among them, aspartylchlorin p6 dimethylester (7b) showed highest phototoxicity against melanoma cells with weakest dark toxicity, which was more phototoxic than verteporfin while with less dark toxicity. It also exhibited better in vivo PDT antitumor efficacy on mice bearing B16-F10 tumor than verteporfin. The biological assays revealed that 7b was localized in multiple subcellular organelles and could cause both cell necrosis and apoptosis after PDT in a dose-dependent manner, resulting in more effective cell destruction. As a result, 7b represents a promising photosensitizer for PDT applications because of its strong absorption in the phototherapeutic window, relatively high singlet oxygen quantum yield, highest dark toxicity/phototoxicity ratio, good water solubility, and excellent in vivo PDT antitumor efficacy.