In the current study, thermoresponsive poly(N-isopropylacrylamide)-doxorubicin (PNIPAM-DOX) hydrogel was synthesized and loaded into pH-responsive poly ethylene glycol)-2,4,6- trimethoxy benzylidene pentaerythritol carbonate (PEG-PTMBPEC) polymersomes in order to fabricate a smart thermo-pH stimuli responsive drug delivery system. Thermo-pH responsive polymersomal formulation of DOX revealed average size of 170 ± 11.2 nm. The prepared system was loaded with PNIPAM-DOX conjugate with encapsulation efficiency of 30.80%. The in vivo release evaluation demonstrated that the DOX release from polymersomal formulation was pH-dependent, i.e. significantly faster drug release at pH 5.5and 6.5 compared to physiological pH. On the other hand, the drug release rate was significantly decreased at 37 °C due to the gelation of PNIPAM-DOX conjugate in the interior compartment of the pH-responsive polymersomes. The in vivo anti-tumor efficiency of the prepared polymersomal formulation of DOX was evaluated implementing C26 tumor-bearing mice after either intravenous (i.v.) or intratumoral (i.t.) single dose injection. The obtained results demonstrated that the prepared system significantly inhibited tumor growth rate in mice receiving single dose via either intravenous or intratumoral injection in comparison with free DOX-treatment group. Furthermore, treatment with polymersomal formulation did not cause any systematic toxicity in terms of pathological alteration of vital organs, survival rate and body weight loss. The prepared smart hydrosomal formulation significantly increased the blood half life time of drug and modified the biodistribution and pharmacokinetic parameters of formulated DOX. In this regard, thermo-pH-dual-stimuli responsive hydrosomal formulation represent a novel approach in nanomedicine development for effective cancer therapy.