This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.