A new series of 2,2'-(9,9-dioctyl-9H-fluorene-2,7-diyl) diacetonitrile-based alternating polyfluorene copolymers (P12-FL, P13-FL, P14-FL) containing ortho-, meta-, and para-substituted phenylene derivatives have been designed, synthesized, and characterized. These polymers were synthesized using a Knoevenagel condensation polymerization reaction and were found to be predominantly in the trans configuration. The resulting polymers were found to be thermally stable and readily soluble in common organic solvents. The structural, configurational, and conformational changes in the backbone caused a wide variation in the absorption and emission maxima of the polymers. The fabricated light-emitting devices showed very good performance in terms of turn-on voltage, electroluminescence, and lifetime properties.