Over the recent decades, conjugated polyelectrolytes have attracted great research interests due to their unique physical and chemical properties. In this feature article, we summarize the recent advances of hyperbranched conjugated polyelectrolytes (HCPEs) in biological sensing and imaging applications. The HCPEs possess three-dimensional core-shell architectures with plenty of functional groups at the peripheries. Through further bioconjugation, the obtained functional HCPEs have wide applications in biomolecules sensing in solution, in vitro cell detection and imaging, specific protein detection in live cells, real-time cell apoptosis detection, gene delivery, and in vivo dual-modal fluorescence and magnetic resonance imaging. Some further outlooks are also highlighted at the end of the feature article.