Esophageal squamous cell carcinoma (ESCC) remains one of the leading causes of cancer-related mortality around the world. The identification of novel serum biomarkers is required for early detection of ESCC. This study was designed to elucidate whether autoantibodies against STIP1 could be a diagnostic biomarker in ESCC. An enzyme-linked immunosorbent assay was performed to detect serum levels of STIP1 autoantibodies in a training cohort (148 ESCC patients and 111 controls) and a validation cohort (60 ESCC patients and 40 controls). Mann-Whitney's U test showed that ESCC patients in two cohorts have higher levels of autoantibodies against STIP1 when compared to controls (P < 0.001). According to receiver operating characteristic analysis, the sensitivity, specificity, and area under the curve (AUC) of autoantibodies against STIP1 in ESCC were 41.9%, 90.1%, and 0.682 in the training cohort and 40.0%, 92.5%, and 0.710 in the validation cohort, respectively. Moreover, detection of autoantibodies against STIP1 could discriminate early-stage ESCC patients from controls, with sensitivity, specificity, and AUC of 35.7%, 90.1%, and 0.684 in the training cohort and 38.5%, 92.5%, and 0.756 in the validation cohort, respectively. Our findings indicated that autoantibodies against STIP1 might be a useful biomarker for early-stage ESCC detection.