have been developed for in vitro and in vivo bioimaging applications. CPDs have attracted considerable attention, because of their potential benefi ts such as relatively facile preparation, high fl uorescence (FL) emission quantum effi ciency, photostability, and low cytotoxicity. [ 2a ] However, CPDs still have some drawbacks, because of the characteristic electronic structures of conjugated polymers. First, in general, π-conjugated polymers have planar geometries and strong intermolecular interactions, because of the extremely stiff and rigid main chains, resulting in highly face-to-face chain packing in the solid state. [ 5 ] Although such an intermolecular π-stacked structure is essential for effective charge-carrier transport in the active layer in thin-fi lm organic optoelectronic devices, [ 6 ] the cofacial packing structure signifi cantly reduces the FL quantum effi ciency in bulk solid fi lms, because it produces crystal domains, and the formed intermolecular excimers have an extremely low transition energy in non-radiative processes. [ 7 ] CPDs are no exception to this rule. [ 2,8 ] Signifi cant FL attenuation has been observed in aqueous colloidal solutions compared with the usual organic solutions. Secondly, although CPDs have much higher photostabilities compared with other fl uorescent materials such as organic dyes [ 9 ] and green fl uorescent In this paper, specifi c molecular design rules are proposed for highly fl uorescent, photostable, conjugated polymer dots (CPDs) applicable for the bioimaging of live cells. CPDs are prepared by nanoprecipitation in water using polydiphenylacetylene (PDPA) derivatives and commercial conjugated polymers. Among these, an amorphous, glassy-state PDPA derivative provides highly porous, coarsened nanoparticles. The nanoparticles are dispersed very well in water, and the polymer chains are either hydrodynamically or thermodynamically stable, with a fully relaxed intramolecular stacked structure. This leads to effective radiative emission decays by restraining collisional quenching and vibrational relaxation to achieve an extremely high fl uorescence (FL) quantum effi ciency. The FL emission quantum yield is as high as 0.76, which is the highest value among those reported for conventional CPDs. The PDPA-based CPD has a very low photobleaching quantum yield (∼10 −9 ), because of its relatively high ionization potential. This aqueous colloidal solution is useful for bioimaging plant and mammalian cells. The excellent FL quantum effi ciency, photostability, and cellular uptake suggest that the present CPD is a very promising probe for bioimaging, particularly for long-term imaging and tracking in live cells or experimental animals.Recently, a wide range of fl uorescent nanoparticles such as inorganic semiconductor quantum dots, [ 1 ] conjugated polymer dots (CPDs), [ 2 ] conjugated polyelectrolyte dots, [ 3 ] and carbon dots [ 4 ]