Electrospun starch acetate (SA) nanofibers with different degrees of substitution (DS) have been developed using formic acid/water as solvents, and their properties and potential applications in drug delivery have been studied. Although SA is biodegradable, biocompatible, and inexpensive, the mechanical properties and potential applications of SA nanofibers have not been studied. This research studied the effect of the solvent system, SA concentration, annealing time, and DS on the morphology and tenacity of SA nanofibers. The water stability of SA nanofibers and drug release profiles using diclofenac as a model drug with the sorption and the dissolution methods have also been investigated. It has been found that annealing not only increased the mechanical properties of SA nanofibers but also led to a low initial burst and a constant release rate. The results also showed that 90% (v/v) formic acid/water solvent system gave even and fine SA nanofibers and the highest tenacity obtained in this study was 17.9 MPa. The SA nanofibers with DS 2.3 retained about 78.0% and 48.0% of its tenacity after 16 and 32 days exposing to 50 degrees C and 90% relative humidity comparing to 77.0% and 40.2% for SA nanofibers with DS 1.1, respectively. In addition, SA nanofibers with DS 2.3 had a lower initial burst and a more constant drug release rate than those with DS 1.1.