In the present research an attempt was made to develop and optimize docetaxel-loaded polyhydroxybutyrate-co-hydroxyvalerate (PHBV) nanoparticles, using modified emulsification solvent evaporation technique and design of experiment (DOE) methodology. Formulation of docetaxel-loaded PHBV nanoparticles was conducted by factor screening studies with Plackett-Burman design (PBD) followed by Box-Behnken experimental design (BBD) to evaluate the effect of independent variables on responses. Five most important independent variables were screened out, which were obtained from failure mode effect analysis (FMEA) and factor screening studies. The effect of formulation parameters on selected responses was depicted by 2-D and 3-D response surface methodology (RSM). The final optimized batch was evaluated by various in vitro characterizations. The observed particle size, zeta potential and entrapment efficiency of optimized formulation was found to be 283 ± 2.79 nm, −17 ± 2.64 mV and 44 ± 0.59% respectively. Morphological studies demonstrated the smooth and spherical shape of nanoparticles. In vitro drug release follows the Peppas-Korsmeyer model of drug release kinetics. Cytotoxicity study was assessed using MCF-7 for percentage inhibition of human breast cancer cell line. These results indicate that the PHBV Nanoparticles could be a promising drug delivery system for efficient prolong drug release.