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One-dimensional negative thermal expansion material has important applications in thermal expansion control and fiber toughened composite. Orthorhombic Al1.6Sc0.4Mo3O12 nanofibers have been prepared by electrospinning. The phase composition, microstructure, morphology, and thermal expansion performance of the samples prepared at various temperatures were investigated. Results show that as-prepared Al1.6Sc0.4Mo3O12 nanofibers are smooth and homogeneous with a fiber diameter of about 370.00 nm, but they show an amorphous structure. Orthorhombic Al1.6Sc0.4Mo3O12 nanofibers can be obtained at 550 ℃ with a diameter of around 140.00 nm. With the increase of post-annealing temperatures, the crystallization of the samples was improved. However, higher post-annealing temperatures will destroy the nanofiber structure of the samples. HTXRD reveals that orthorhombic Al1.6Sc0.4Mo3O12 nanofibers show anisotropic NTE in 25–700 °C. The expansion coefficients αa, αb, and αc of Al1.6Sc0.4Mo3O12 nanofibers are −5.05 × 10–6 °C−1, 4.41 × 10–6 °C−1, and −3.18 × 10–6 °C−1, respectively. The αv is −3.83 × 10–6 °C−1, and the corresponding αl is −1.28 × 10–6 °C−1. [ABSTRACT FROM AUTHOR]