In recent years, there has been considerable interest in developing biodegradable polymer based nanoparticles to effectively deliver a drug to a target site. Polymeric materials used for preparing nanoparticles for drug delivery must be biocompatible and preferably biodegradable. The objective of this study was to fabricate nanoparticles from acetylated dioscorea starch having particle size less than 200 nm with minimum polydispersity index (PDI). Acetylated starch was synthesized by reacting native dioscorea starch with acetic anhydride in the presence of sodium hydroxide as a catalyst. Acetylated starch nanoparticles were then fabricated by spontaneous emulsification and solvent evaporation method under high speed homogenization. A systematic investigation on independent variables revealed that solvent type, surfactant type and concentration, homogenization speed and time have significant influence on the size and polydispersity of nanoparticles. Hence, response surface methodology based on central composite design was employed to fabricate the desired nanoparticles (particle size less than 200 nm and PDI < 0.2) under optimized condition (ethyl acetate, 1.45% of Pluronic (R) F127, homogenization speed of similar to 16,000 rpm and homogenization time of 18.25 min).