In vivo fluorescence imaging within the second near-infrared region (NIR-II, 1000-1700 nm) has advantages of a higher signal-to-background ratio (SBR), spatial resolution, and deeper tissue penetration depth than that in the visible (400-650 nm) and the first near-infrared window (NIR-I, 650-1000 nm). Here, we have synthesized three NIR-II fluorescent polymer dots (P1-Pdots, P2-Pdots, and P3-Pdots) for the NIR-II imaging. These Pdots were designed and optimized by using benzodithiophene as a donor unit and quinoxaline derivatives as acceptor units. The backbone and side chains of the quinoxaline acceptor units were varied to optimize the fluorescence performance. We found that the substituted position of alkoxy groups in the side chains plays an important role in enhancing the NIR-II window quantum yield (QY). In one case, the resulting nanoparticles (P1-Pdots) exhibited an emission peak at similar to 1100 nm and a high QY of similar to 1%. P1-Pdots possesses additional advantages for bioimaging, including deep tissue penetration depth, good stability, and biocompatibility. The blood vessel imaging of the mouse by P1-Pdots could be clearly observed with high spatial resolution and displayed an SBR of similar to 2.1. Besides, PI-Pdots has further demonstrated its applications for tumor imaging of tumor-bearing nude mice, such as assessing the in vivo angiography and monitoring tumor vasculatures. Our results indicate the Pdots afford high fluorescence signals and spatial resolution for imaging deep tissues.