Photodynamic therapy (PDT) is an attractive therapeutic modality with high therapeutic efficacy and less side effects compared with other therapies. Herein, we developed a series of cyclometalated Ir III complexes based on 2,2′-biimidazole with different alkyl substitutions (methyl, ethyl, propyl, and butyl). These complexes can efficiently produce singlet oxygen upon light irradiation. In vitro photocytotoxicity towards four cell lines (HeLa, A549, A549R, and LO2) was examined. Ir1-Ir5 show quick penetration of cells, remarkable mitochondrial accumulation, strong phototoxicity, and they exhibit high selectivity