We explore the interactions between a fluorescein (FAM)-labeled single-stranded DNA (P), graphene oxide (GO), and a cationic conjugated polymer, poly [(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP). It is found that the fluorescence change of P-GO-PFP system is dependent on the addition order of P and PFP. When adding PFP into P/GO complex, the fluorescence resonance energy transfer (FRET) from PFP to P is inefficient. If P is added to PFP/GO complex, efficient FRET is obtained. This may be attributed to the equal binding ability for P and PFP to GO. The results of time-resolved fluorescence and fluorescence anisotropy support the different fluorescent response under different addition order of P and PFP to GO. Based on the above phenomenon, we demonstrate a method to reduce the high background signal of a traditional PFP-based DNA sensor by introducing GO. In comparison to the use of single PFP, the combination of PFP with GO-based method shows enhanced sensitivity with a detection limit as low as 40 pM for target DNA detection.