We prepared three optically active polyfluorenes (PFs), poly[2,7-{9,9-bis[(S)-2-methyloctyl]}fluorene] (S1) and poly[2,7-{9,9-bis[(S)- and/or (R)-3,7-dimethyloctyl]}fluorene] (S2 and/or R2) and an optically inactive poly [2,7-(9,9-di-n-decyl)fluorene] (3) in order to investigate the alkyl side-chain effects on their (chir)optical properties in both solution and solid states. PF S1 showed more intense circular dichroism (CD) signals in THF solution at higher and lower temperatures due to a stiffer backbone structure. Although the CD and UV-visible spectra suggested that similar thermodriven order-disorder conformational transitions occurred for all of these PFs, the highly ordered conformations formed upon cooling showed less planar structures with the stereocenters closer to the PF backbones, whose planarity decreased in the order of 3, S2/R2, and S1. In the pristine spin-coated films on quartz, with the stereocenters closer to the PF backbones, the corresponding PFs showed more intense bisignate CD signals and blueshifts in the emission peaks.