Conjugated polymers have been widely used in optical sensors, light-emitting diodes and solar cells, due to their attractive optical and semiconducting properties. It is widely accepted that the optical and electrical properties of conjugated polymer molecules depend on the conjugated segments, i.e., chromophores in conjugated polymer molecule. The study of the evolution of the absorption and emission properties of single conjugated polymer molecules is essential to provide complementary information for the influence of conformation of conjugated polymer on its energy transfer process, as well as on the performance of optoelectronic devices based on conjugated polymers. Although the extensive studies have been reported to elucidate the optical properties of conjugated polymers with single molecule spectroscopy, simultaneous revealing their absorption and emission properties and their real-time evolution are rarely reported. In this paper, we simultaneously measure the absorption and emission properties of chromophores in single Poly[2,7-(9,9-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole](PFO-DBT) conjugated polymer molecules and their real-time evolution by frequency-domain reconstructed defocused wide-field imaging. The emission dipole orientation of chromophore is achieved by applying defocused wide-field fluorescence imaging. The change of defocused patterns of individual polymer chain describes the angular distribution of emitted light and thus the emitting dipole orientation. Meanwhile, the absorption dipole orientation of chromophore in single conjugated PFO-DBT polymer molecule can be clarified in reconstructed frequency-domain imaging by modulating the relative phase of the pulse pairs and performing Fourier transform to the photoluminescence response. The population density of excited state of absorbing chromophore depends both on the relative phase between the ultrashort pulse pairs and on the orientation of absorption transition dipole moment of the chromophore. By extracting the frequency-domain information of fluorescence that is proportional to the population density of excited state, the evolution of absorption dipole orientation of chromophore can be derived. We distinguish three cases for the evolution of chromophores of single PFO-DBT conjugated polymer molecules: the absorption and emission chromophores both keep constant in single PFO-DBT conjugated polymer molecules; one of the dipole orientations of absorption and emission changes, while the other remains unchanged; both of them change simultaneously. The results may pave the way for the further understanding of the role of conformation in the energy transfer pathway in both natural and artificial light harvesting systems at nano- and micro-level.