An aligned polyacetylene (PA) film was synthesized in a macroscopically-aligned nematic liquid crystal (N-LC) solvent using a gravity-flow method. Long and single nanofibers of less than 100 nm in radius were successfully prepared by ultrasonication of the aligned PA film immersed in ethanol. The usual PA film was synthesized in an isotropic solvent, such as toluene, only yielding short and non-dispersed fibers after the ultrasonication due to the entangled fibril morphology. Entanglement-free fibrils of the aligned PA were well-separated into single fibrils through ultrasonication, even without a surfactant.