Background Tumor mutational burden (TMB) has both prognostic value in resected non-small cell lung cancer (NSCLC) patients and predictive value for immunotherapy response. However, TMB evaluation by whole-exome sequencing (WES) is expensive and time-consuming, hampering its application in clinical practice. In our study, we aimed to construct a mutational burden estimation model, with a small set of genes, that could precisely estimate WES-TMB and, at the same time, has prognostic and predictive value for NSCLC patients. Methods TMB estimation model was trained based on genomic data from 1056 NSCLC samples from The Cancer Genome Atlas (TCGA). Validation was performed using three independent cohorts, including Rizvi cohort and our own Asian cohorts, including 89 early-stage andnlate-stage Asian NSCLC patients, respectively. TCGA data were obtained on September 3, 2018. The two Asian cohort studies were performed from September 1, 2018, to March 5, 2019. Pearson's correlation coefficient was used to assess the performance of estimated TMB with WES-TMB. The Kaplan-Meier survival analysis was applied to evaluate the association of estimated TMB with disease-free survival (DFS), overall survival (OS), and response to anti-programmed death-1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) therapy. Results The estimation model, consisted of only 23 genes, correlated well with WES-TMB both in the training set of TCGA cohort and validation set of Rizvi cohort and our own Asian cohort. Estimated TMB by the 23-gene panel was significantly associated with DFS and OS in patients with early-stage NSCLC and could serve as a predictive biomarker for anti-PD-1 and anti-PD-L1 treatment response. Conclusions The 23-gene panel, instead of WES or the currently used panel-based methods, could be used to assess the WES-TMB with a high relevance. This customized targeted sequencing panel could be easily applied into clinical practice to predict the immunotherapy response and prognosis of NSCLC.