Using Suzuki and Yamamoto coupling reactions, copoly-(9,9-dioctylfluorenes) (CPF) were synthesized and compared regarding their photophysical properties using the spectroscopic and ab initio DFT approaches. The CPFs were functionalized by benzo [2,3,5] thiadiazole (BT) or carbazole-3,6-diyl (3,6-Cz). The latter was used to introduce different luminophore fragments, including Nile red and 4-pyrrolidinyl-1,8-naphthalimide derivatives. The effect of the two synthesis techniques on the polymer microstructure, the influence of embedding of 3,6-Cz moieties in the polymer backbone on polymer structuring, and the impact of the end groups like novel quinoxaline-containing compounds on the luminescent properties of CPFs were investigated. By comparing electron density distribution using the ab initio DFT approach with photoluminescence, it was shown that Suzuki reaction provides a chain microstructure with individual BT fragments separated by 9,9-dioctylfluorene monomeric units, while Yamamoto reaction leads to the blocks of BT units. This effect leads to different CPF photophysical properties (absorption and emission spectra).