Novel poly(9,9-dioctlylfluorene) (PFO) nanofibers were fabricated by solution template wetting of anodic aluminum oxide (AAO) templates with a pore diameter of 25 nm. Individual nanofibers displayed a pronounced axially polarized luminescence with a typical emission dichroic ratio of 15 and low spread of the emissive species angular distribution. The strong optical characteristics were ascribed to intrachain reorientation of amorphous PFO to a more planar and elongated beta-phase conformation induced by mechanical strain during polymer template pore infiltration. Absorption optical spectroscopy on nanofiber mats confirmed formation of 24% beta-phase emissive segments, which dominated the nanofiber luminescence characteristics. X-ray diffraction measurements were used to confirm and quantify the extent of nanofiber internal molecular alignment.