The proven benefit of third-line treatment in advanced non-small cell lung cancer (NSCLC) is still unclear. We retrospectively evaluated the outcome of advanced NSCLC patients treated with third-line therapies in our institution, especially focusing on efficacy and toxicity between mono-therapy and doublets chemotherapy, aiming to assess the value of third-line treatment and evaluate the efficacy of different regimens in third-line treatment. Three hundred and seventeen patients received a second-line treatment among 620 advanced NSCLC after failure of first-line chemotherapy. One hundred and twenty-six patients from this group were offered third-line or further-line treatments. Survival analysis was conducted based on Kaplan-Meier method, and Chi-square was used to compare data between second-line and third-line group. There were significant differences in overall survival between second-line and third-line treatments (17.70 months vs. 24.03 months, P < 0.0001). Twenty-four patients received single chemotherapy, epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) were used to treat thirty-three patients and sixty-nine patients received doublet chemotherapy in third-line treatment. The progression-free survival (PFS) after third-line therapy was 2.37 months in all patients and 2.30 months, 2.80 months, 2.97 months, in the doublet chemotherapy, single-agent chemotherapy, and EGFR-TKIs arms, respectively (P = 0.033). Cancer-related symptom relief improvement was confirmed in 78.9% patients (60/78). Forty-eight patients had no symptoms as confirmed by imaging examination. Patients with advanced NSCLC could get benefits from third-line treatments. Those patients could obtain a moderate progression-free survival and conspicuous improvement in the cancer-related symptom. Mono-therapy was recommended in third-line treatment.