Fluorescence imaging at longer wavelength and autofluorescence-free imaging were two main strategies to improve the optical imaging performance of traditional near-infrared (NIR) fluorescence imaging, and the design of corresponding imaging agents were heavily explored recently. Herein, polymer-blend dots (PBdots) with strong fluorescence and afterglow luminescence in both NIR-I (700–900 nm) and NIR-II (900–1700 nm) windows were successfully fabricated by encapsulating two functional semiconducting polymers into one nanoparticle. With efficient intra-particle interactions, the PBdots exhibited an afterglow lifetime of 181 s after 730 nm NIR laser activation, which can be activated in vivo. The obtained PBdots with similar emission intensities in NIR-I and NIR-II regions enable fair comparison between different optical imaging modalities, which provides a clear guidance for the choice of suitable imaging modality for different purpose. As the first imaging agents which integrate NIR-II fluorescence imaging and NIR-I afterglow imaging, the PBdots also provide both high resolution and large signal-to-background ratio for blood vessel, lymph node and tumor imaging in living mice.