Background: EC-CRT-001 is a phase II trial investigating the efficacy of toripalimab (an anti-PD1 antibody) combined with definitive chemoradiotherapy (CRT) in locally advanced esophageal squamous cell carcinoma (ESCC) and the results demonstrated encouraging antitumor activity and acceptable toxicity. This exploratory analysis was performed to evaluate the role of circulating tumor DNA (ctDNA) and blood-based tumor mutational burden (bTMB) for efficacy prediction. Methods: Forty-two patients from the phase II trial (NCT04005170) who received toripalimab (240 mg every 3 weeks for up to 1 year, or disease progression, or unacceptable toxicity) combined with definitive CRT (irradiation with 50.4 Gy in 28 fractions concurrent with 5 cycles of weekly paclitaxel in 50 mg/m² and cisplatin in 25 mg/m²) were enrolled. Plasma samples were collected before, during, and after CRT. Targeted next-generation sequencing was performed on a total of 118 plasma samples and 35 matched tumor samples using a panel covering 474 cancer-associated genes. Results: Twenty-six (62%) of 42 patients achieved clinical complete response (cCR) at three months after CRT. ctDNA was detected in 29 (72.5%) of 40 patients at baseline. The positive rate of ctDNA decreased to 43.9% (20/41) during CRT and continued to descend to 27.0% (10/37) at the completion of CRT. A higher cCR rate was observed in patients with negative during-CRT ctDNA compared to those with detectable ctDNA (82.6% vs. 38.9%, P = 0.008). Patients with post-CRT detectable ctDNA also had poorer cCR rate (30.0% vs. 77.8%, P = 0.017). Within a median follow-up of 24.0 months (IQR 13.7-27.9), the median progression-free survival (PFS) was 12.2 months (95%CI: 8.4-16.0) and median overall survival (OS) was not reached for the whole cohort. Patients with during-CRT detectable ctDNA had shorter PFS compared to ctDNA-negative patients (HR = 2.57, 95%CI: 1.18-5.60, P = 0.014). Similarly, patients with post-CRT detectable ctDNA also had a significantly increased risk of disease progression (HR = 2.88, 95%CI: 1.21-6.83, P = 0.012) and death (HR = 3.67, 95%CI: 1.41-9.55, P = 0.004). Moreover, patients with higher bTMB ( > 1) detected during CRT were associated with a favorable OS (HR = 0.33, 95%CI: 0.13-0.88, P = 0.027). Improved PFS was also observed in those patients with higher post-CRT bTMB ( > 3) (HR = 0.28, 95%CI: 0.08-0.96, P = 0.042). Conclusions: Negative ctDNA status and higher bTMB during or after CRT were associated with better tumor response and favorable survival in ESCC patients who underwent definitive CRT combined with toripalimab. Dynamic ctDNA has great potential in predicting efficacy and risk of disease progression in ESCC. Clinical trial information: NCT04005170.