The third-order nonlinear optical (NLO) susceptibility for morphologically controlled polydiacetylene (PDA) nanocrystals (NCs) and PDA nanofibers (NFs) have been determined for the first time by the experimental combination of transient pump-probe spectroscopy and spectroscopic ellipsometry. The figure of the merit of PDA NFs was much superior to PDA NCs and/or PDA bulk crystals, and the excitonic relaxation time was of order of sub-pico second. Namely, this is the first case to reveal the morphological effect on NLO response. PDA NFs having the long effective π-conjugation length are one of the most promising organic third-order NLO nanomaterials toward the photonic device application.