Drug resistance has become a main obstacle for the effective treatment of lung cancer. To address this problem, a novel biocompatible nanoscale package, poly(D,L-lactide-co-glycolide)-Tween 80, was designed and synthesized to overcome paclitaxel (PTX) resistance in a PTX-resistant human lung cancer cell line. The poly(D,L-lactide-co-glycolide) (PLGA)-Tween 80 nanoparticles (NPs) could efficiently load PTX and release the drug gradually. There was an increased level of uptake of PLGA-Tween 80 in PTX-resistant lung cancer cell line A549/T, which achieved a significantly higher level of cytotoxicity than both PLGA NP formulation and Taxol (R). The in vivo antitumor efficacy also showed that PLGA-Tween 80 NP was more effective than Taxol (R), indicating that PLGA-Tween 80 copolymer was a promising carrier for PTX in resistant lung cancer.