Imaging-guided photodynamic therapy (PDT) represents an advantage in cancer therapy because of its optically controlled selectivity, negligible side effects, and ability to monitor the therapeutic effect in situ in real time. However, the photosensitizers currently in use have poor water solubility and an inability to target tumors, whereas semiconductor quantum dots and metal-based complexes that have excellent water dispersity have the disadvantage of exerting a potential toxicity because of the heavy metals. Therefore, this work describes the preparation of the red fluorescent N,S-doped carbon dots (N,S-CDs) from a polythiophene derivate that generate singlet oxygen ( 1 O 2 ) with a quantum yield of ∼8% under light irradiation. Moreover, these CDs possess the ability to penetrate tumor cells and mainly accumulate into the lysosome and mitochondria, as well as excellent biocompatibility, chemostability, and a large Stokes shift (215 nm). Hence, the N,S-CDs may be used as a potential effective photosensitizer for imaging-guided PDT.