We report here the design and synthesis of a series of π-conjugated fluorescent dyes with D-A-D (D: donor; A: Acceptor), D-π-D, A-π-A, and D-π-A for applications as the signaling motif in biologicalsynthetic hybrid foldamers for DNA detection. Horner-Wadsworth-Emmons (HWE) reaction and Knoevenagel condensation were demonstrated as the optimum ways for construction of long π-conjugated systems. Such rod-like chromophores have distinct advantages, as their fluorescence properties are not quenched by the presence of DNA. To be incorporated into the backbone of DNA, the chromophores need to be reasonably soluble in organic solvent for solid-phase synthesis, and therefore a strategy of using flexible tetra(ethylene glycol) (TEG) linkers at either end of these rodlike dyes were developed. The presence of TEG facilitates the protection of the chain-growing hydroxyl group with DMTrCl (dimethoxy trityl chloride) as well as the activation of the coupling step with phosphoramidite chemistry on an automated DNA synthesizer. To form fluorescence resonance energy transfer (FRET) pairs, six synthetic chromophores with blue to red fluorescence have been developed and those with orthogonal fluorescent emission were chosen for incorporation into DNA-chromophore hybrid foldamers.