The discovery of novel photosensitizers with potent phototoxicity and desirable water solubility is an urgent task for photodynamic therapy. Herein, a series of amino acid-modified aza-BODIPY photosensitizers were synthesized and evaluated. These new PSs exhibited enhanced aqueous solubility, increased 1O2 generation efficiency, and an improved photo-dark toxicity ratio. Aspartic acid-modified PS of 1a, which possessed intense NIR absorption and high 1O2 quantum yield, demonstrated the most potent efficacy toward the investigated tumor cell lines without using an emulsifier. Subcellular localization, cell-based ROS production, and cell death pathway of 1a were studied. In vivo fluorescence imaging and ex vivo organ distribution assays manifested that 1a possessed reasonable distribution and clearance. In vivo PDT studies indicated that 1a revealed advantages over Ce6 and our previously optimized PS of BDP-4. It not only afforded an excellent PDT effect with a low drug dose under only single-time photoirradiation but also induced an antitumor immunological response.