The nature of chain aggregation in solution always results in variable spin-coated film mesoscale morphology and uncontrollable device performance. The abundant variety and increasing chemical complexity of conjugated polymers induced additional diverse electrostatic and dispersion interactions (non-covalent interactions), although it is not fully understood how the interplay of these forces results in the observed conformational order, chain aggregates and film morphologies. Herein, we present a precise study on the role of non-covalent interaction in the self-organization behavior, conformational order and optoelectrical properties of polyfluorene (PPFOH) toward tuning its electroluminescence (EL). The supramolecular PPFOH system consisted of an intrinsically doped hydrogen-bond-assisted microstructure as a “guest” and a blue light-emitting backbone chain as a “host”, which show a special binary emissive property of solution-induced self-dopant formation in the amorphous films. As a result of a strong non-covalent interaction between polymer chains and solvent molecules (type II solvent), a likely distorted or fold chain in rod-coil or branch cluster shows a narrow and strong aggregation emission at 525–540 nm. Low-polar solvents (called type I) can also induce a shoulder low-energy emission at 550–580 nm in the films, attributed to the extended and stretched chain complex for the tendency of interchain hydrogen-bonding interaction. Further evidence from nanoscale infrared (AFM-IR) analysis confirmed the stronger hydrogen-bonding interaction in the type II films than those in the type I films. Finally, supramolecular PPFOH electroluminescence colours can be tuned from blue to sky blue, green, white, yellow and orange.