By introducing a naphthothiadiazole (NT) unit as the main building block, a non-doped and red emissive conjugated polymer poly(9,9-dihexylfluorene- alt -naphthothiadiazole) (PFNT) is readily obtained through a two-step synthesis. Since the NT unit has a large twist angle with its neighboring segment, the aggregation-induced quenching (AIQ) effect of PFNT can be effectively suppressed in the condensed state. As a result, the corresponding PFNT polymer dot (Pdot) exhibits a high fluorescence quantum yield of 53.2% with peak emission at 616 nm, which is one of the most efficient red Pdots known. PFNT Pdot shows good biocompatibility and can be employed for living cell fluorescent imaging with high brightness. It also can be used for specific subcellular organelle imaging through immunofluorescence labeling. Furthermore, the PFNT Pdot demonstrates much better photostability for long-time cell fluorescence imaging than commercial red dyes. The high performances of PFNT Pdot make it a promising fluorescent probe for practical bioapplications.& COPY; 2023 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.