Docetaxel (DTX) is one of the most effective antineoplastic drugs. However, its current clinical administration, formulated in tween80, causes serious side effects. This study is focused on preparation and evaluation of poly(lactide-co-glycolide) nanoparticles (NPs) containing DTX to remove tween80. Drug encapsulation efficiency, in-vitro drug release, cellular cytotoxicity, and in-vivo biodistribution of NPs in mice after intravenous administration were investigated. The average diameter of the NPs was approximately 172-178 nm with encapsulation efficiency of 68%. A burst release of approximately 30% (w/w) of the loaded drug followed by a sustained release profile was observed. Cellular mortality of the NPs was more than or at least as great as DTX free drug; for example, cell viability measured at 100 nmol/l drug concentration was decreased from 50.9% for DTX free drug to 15.9% for the NP formulation after 48 h incubation with T47D cells. The DTX plasma amount remained at a good level (13% of the initial dose) in the NP formulation compared with the DTX conventional formulation, which is approximately 0.5% of the initial dose, was present in plasma up to 2 h. Poly(lactide-co-glycolide) NPs containing DTX prepared in this study may be regarded as a suitable and superior formulation for the current formulation in the market containing tween80 with improved cancerous cell mortality and biodistribution characteristics.