Amorphous poly(9,9-di-n-octyl-2,7-fluorene)(PFO) thin films were characterized in situ via thermal annealing based on grazing incidence X-ray diffraction(GIXRD) profiles, UV-visible absorption spectrophotometry, and Fourier transform infrared spectroscopy(FTIR). The results of GIXRD indicated that the amorphous phase transformed into a crystalline phase when the annealing temperature was higher than 80 °C. Different outcomes were elicited for the intensities and d-spacings of the diffraction peaks below and above 80 °C, which were attributed to the formation of the κ-phase. The mechanism of phase transition was revealed by in situ UV-visible absorption and FTIR spectra, whereby the rearrangement of the side chains was dominant and the movement of the main chains was minimal, even when the annealing temperature was lower than 80 °C. In contrast, the rearrangement of the main chains was dominant when the temperature was higher than 80 °C.