Drug resistance remains the key factor that results in the high mortality of lung cancer patients. Therefore, it is very important to explore the mechanisms underlying the lung cancer drug resistance by establishing a third-generation molecular targeting drug-resistant cell line of non-small cell lung cancer (NSCLC). This study successfully established an Osimertinib-resistant cell line, HCC827OR, based on HCC827 cell line through combining the methods of intermittent high-dose selection and concentration-increasing. Cytotoxicity of Osimertinib against cancer cells was firstly evaluated through using the CCK8 assay kit, and resistance index (RI) was used to evaluate the sensitivity of cancer cells to Osimertinib; morphological changes of the cancers cells before and after acquiring drug resistance were stained and imaged using an optical microscopy. Western blot was used to detect the expression level of epidermal growth factor receptor (EGFR) and its downstream signaling proteins; mutation of EGFR was measured through using the next generation sequencing (NGS); cell cycle and scratch assays were performed to evaluate cell proliferation and migration ability. The RI of HCC827OR was calculated to be 18.65, demonstrating that HCC827OR was Osimertinib-resistant cells. Compared with the wild-type cells, Osimertinib-resistant cells showed a greater karyoplasmic ratio. Additionally, reduced expression of the p-EGFR and increased expression of cell proliferation-associated p-AKT indicated that the HCC827OR probably acquired the Osimertinib-resistant character through a non-EGFR-dependent signaling pathway. Meanwhile, flow cytometry and scratch assay results demonstrated that HCC827OR cells have significant G2/M cycle arrest and enhanced migration ability in comparison with the HCC827. This study successfully established an Osimertinib-resistant NSCLC cell line, and tentatively demonstrated the acquisition of drug-resistant characteristics by the HCC827OR through a non-EGFR-dependent pathway, thus providing an important in vitro model for lung cancer drug-resistance research.