Background: Patients with esophageal cancer benefited from neoadjuvant chemotherapy combined with immunotherapy. However, the sequence of administration during neoadjuvant therapy remains under considerable debate. Methods: A study on 23 enrolled patients with esophageal cancer who received 2 cycles of neoadjuvant treatment of dose-limiting chemotherapy combined with sequential pembrolizumab and 2 cycles of adjuvant treatment of dose-limiting chemotherapy combined with sequential pembrolizumab was carried out. Serial plasma samples were collected and subjected to T cell receptor (TCR) sequencing. Tissues with pre- and post-neoadjuvant therapy were obtained and performed with whole-exome sequencing and transcriptome sequencing. Results: After neoadjuvant therapy, the percentage of patients with a pathological major response (MPR) was 39.1% (9/23), consisting of 21.7% (5/23) completed pathological response (cPR). TCR sequencing analysis showed that the peripheral blood-dominated TCR clones significantly augmented during the neoadjuvant treatment. Tumor mutational burden (TMB) was higher in the MPR group than in the non-MPR group at baseline. In addition, infiltrating CD8+ T cells increased and regulatory T (Treg) cells decreased in the MPR group after neoadjuvant therapy. While Treg cells increased in the non-MPR group after neoadjuvant treatment. Conclusions: Dominated TCR clones increased during the neoadjuvant limited-dose chemotherapy stage, which would contribute to sequential PD-1 blockade. Additionally, TMB, CD8+ T cells, and Treg cells are associated with clinical benefits during treatment.