Temperature-sensitive liposomes (TS-liposomes) have been studied for chemotherapeutic purposes to enhance the release of anticancer drugs at tumor sites. In this study, we prepared poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethylene glycol (PEG)-modified TS-liposomes (PETS-liposomes). PETS-liposomes significantly increased in vitro drug release in serum compared with PEG-fixed or PNIPAM-AAM-modified liposomes. Furthermore, incorporation of both PNIPAM-AAM and PEG into PETS-liposomes enhanced the stabilities of liposomes in serum by inhibiting protein adsorption. In addition, to investigate the therapeutic efficacy of doxorubicin (DOX)-loaded PETS-liposomes, the in vivo antitumor activity of liposomes in combination with hyperthermia was evaluated in a B16F10 melanoma tumor-bearing mouse model. PETS-liposomes showed much higher levels of tumor growth inhibition than PEG-fixed or PNIPAM-AAM-modified TS-liposomes. Moreover, the antitumor activity of PETS-liposomes was enhanced significantly when they were administered in combination with hyperthermia. PETS-liposomes were found to be highly efficacious carriers for the in vivo delivery of anticancer drugs, and to have potential anticancer applications in combination with hyperthermia.