Background: Patients with non-small cell lung cancer (NSCLC) can benefit from the treatment of preoperative neoadjuvant chemotherapy combined with immunotherapy. However, the selection in concurrent or sequential neoadjuvant treatment is still controversial. Therefore, the effective form of sequential neoadjuvant treatment as well as predictive biomarkers are still need to be further investigated. Methods: We carried out a multi-omics study on 12 enrolled NSCLC patients who received sequential neoadjuvant and adjuvant treatments. The responses to therapy were rigorously evaluated. Biopsies pre and post neoadjuvant therapy were performed with whole-exome sequencing, transcriptome sequencing. Plasma at the different timepoints during therapy were collected and sequenced with TCR sequencing and ctDNA sequencing. Results: After neoadjuvant therapy, 10 were major pathological responses (MPR) and 2 were non major pathological responses (non-MPR). The analysis of TCR sequencing showed that high diversity along with the increase of dominance of T-cell receptor repertoire were observed after chemotherapy. At baseline, tumor mutational burden (TMB) and tumor neoantigen burden (TNB) were higher in MPR group when compared with non-MPR group. Furthermore, tumor infiltrating CD8+ naïve T cells, Tregs and monocytes were associated with clinical benefit and patients with high ratio of CD8+ naïve T cells and monocytes have better pathological response. Conclusions: In this study, sequential neoadjuvant treatment of dose-limiting chemotherapy combined with sintilimab showed a promising antitumor activity in patients with resectable NSCLC. The changes of genomic, transcriptome and TCR repertoire were comprehensively analyzed and correlated with clinical outcomes. TMB, TNB and TCR repertoire are associated with the clinical benefit. The sequential therapy would also impact the immune microenvironment. These result indicate that sequential neoadjuvant treatment of chemotherapy combined with immunotherapy could be a potential treatment for a defined group of patients with NSCLC.