Three ortho-, meta-, and para-linked polymers derived from 9,9-dioctylfluorene (FO) and dimethoxyl-biphenyl (DMBP) were designed and synthesized via catalyst-transfer Suzuki coupling polymerization with palladium(0) catalyst as initiator. Compared to PFO, the conjugated polymers of PFO-o-DMBP, PFO-m-DMBP, and PFO-p-DMBP displayed a significantly blued-shifted absorption and emission spectra with the change of the connecting site from ortho-, meta- to para-position, and the varying degrees were ascribed to the different types of steric hindrance of ortho-, meta-, and para-linkage, which partly hinder the intermonomer rotation of the polymer backbone, giving rise to molecular configuration from linear, zigzag to intertwined structure, and resulting in shortened conjugation length. The optical bandgap calculated from the onset of absorption spectra of the three polymers in solid film are all wider than that of the PFO, indicating that the incorporating of dimethoxyl-biphenyl increased the chain-twisting hindrance and influenced the molecular conformation of the copolymer. Systematical investigation of electrochemical and photophysical properties of the conjugated polymers suggests that the incorporation of dimethoxyl-biphenyl via ortho-, meta-, and para-linkage is an efficient and economic way to modify the properties of polyfluorenes.