The effect of a nonionic surfactant on various energy-transfer and quenching processes in deoxyribonucleic acid (DNA) sequence detection using the cationic conjugated polymer (CCP) poly( [9,9-bis(N,N,N-trimethylammonium)hexyifluorene]-co-1,4-phenylene diiodide) and peptide nucleic acid labeled with fluorescein (PNA-Flu*) has been investigated. Steady-state and time-resolved measurements reveal that nonionic surfactant enhances both sensitivity and selectivity in targeted DNA sequence detection. The enhancement of the sensitivity is ascribed to an increase in the CCP quantum yield due to the surfactant breaking up polymer aggregates, increasing the isolated polymer chain concentration, and reducing interchain quenching. This also increases the polymer surface-to-volume ratio. The sensitivity is also improved as the surfactant reduces CCP quenching by DNA and buffer solution through incorporation of the CCP in micelles, which reduces charge-transfer processes. Enhancement of the selectivity is achieved through the reduction of the hydrophobic interaction between the CCP and free PNA-Flu*. Time-resolved fluorescence lifetime analysis provides an effective way to differentiate these particular effects in the DNA-PNA-Flu assays. Comparing the decay profiles of the CCIP, CCP/DNA, and CCIP/complementary DNA:PNA-Flu*, it was found that both surfactant and DNA do not alter dramatically the isolated polymer chain lifetime (the first increases the concentration of the single chains by breaking up the polymer aggregation and the latter reduces the concentration of the isolated polymer chain by static quenching), therefore the change in the isolated polymer chain fluorescence lifetime can predominantly reflect the quenching of the donor by FRET to the PNA fluorescent label. Here, surfactant increases Forster radius by enhancing the polymer fluorescence quantum yield. Furthermore, surfactant also increases the difference in the donor lifetimes between the complementary and noncomplementary cases. This difference reflects the increase in the energy-transfer efficiency and hence higher selectivity. Fluorescein quenching is mediated by the CCP which causes Flu*-Flu* self-quenching via a PNA-CCP hydrophobic interaction. Optimization or minimization of various interactions to achieve the best DNA detection sensor is discussed.