Fluorescent conjugated polymers (CPs) have attracted considerable interest in biosensing owing to their high fluorescence, tunable bandgap, and good biocompatibility. Aiming at acquiring the desired optical responses of CPs for bioapplications, it is essential that the CPs bind to biological targets with high efficacy and affinity. However, the efficient binding of CPs is largely driven by their effective interaction with target surfaces. In this Review, we will focus on the different surface interactions that pervade between CPs and biological targets. The multiple surface interactions can lead to changes in spatial conformation and distribution of CPs, which manifest alterable optical properties of CPs based on accumulation of target-directed CPs, Forster resonance energy transfer mechanism, and metal-enhanced fluorescence mechanism. Then, we display diverse bioapplications applying CPs-based surface interactions, such as cell imaging, imaging-guided detection, and photodynamic therapy. Finally, the challenges and future developments to control the efficient attachment of CPs to biological targets are discussed. We expect that the understanding of surface interactions between CPs and biological targets benefits the CPs-based system design and expands their applications in biological detections and therapies.