Two types of cationic polyfluorene copolymers (FHQ, FPQ) with a same π-conjugated structure but different counterions (bromide (BR), tetraphenylborate (PB)) were synthesized and studied as a fluorescence resonance energy transfer (FRET) donor to fluorescein-labeled DNA (ssDNA-Fl). The counterions accompanying the polymer chain for charge compensation are expected to perturb complexation with DNA and modify the fine-structure of D/A complex on molecular scale, which may influence the competition between the desirable FRET and energy-wasting charge transfer quenching. The PL quenching constant of ssDNA-Fl by Stern−Volmer plot was significantly reduced in the presence of the polymers with tetraphenylborate (4.3 × 10⁶ M⁻¹ for FHQ-BR vs 2.2 × 10⁶ M⁻¹ for FHQ-PB, 2.8 × 10⁶ M⁻¹ for FPQ-BR vs 1.3 × 10⁶ M⁻¹ for FPQ-PB). The resulting FRET-induced signal was amplified 2 to 8.6 times by exchanging bromide with tetraphenylborate as a counterion, suggesting a simple way for kinetic control of energy transfer to maximize signal amplification in conjugated polymer-based FRET biosensors.