AbstractEsophageal squamous cell carcinoma (ESCC) is a highly prevalent and aggressive malignancy, and timely diagnosis of ESCC contributes to an increased cancer survival rate. However, current detection methods for ESCC mainly rely on endoscopic examination, limited by a relatively low participation rate. Herein, ferric‐particle‐enhanced laser desorption/ionization mass spectrometry (FPELDI MS) is utilized to record the serum metabolic fingerprints (SMFs) from a retrospective cohort (523 non‐ESCC participants and 462 ESCC patients) to build diagnostic models toward ESCC. The PFELDI MS achieved high speed (≈30 s per sample), desirable reproducibility (coefficients of variation < 15%), and high throughput (985 samples with ≈124 200 data points for each spectrum). Desirable diagnostic performance with area‐under‐the‐curves (AUCs) of 0.925–0.966 is obtained through machine learning of SMFs. Further, a metabolic biomarker panel is constructed, exhibiting superior diagnostic sensitivity (72.2–79.4%, p < 0.05) as compared with clinical protein biomarker tests (4.3–22.9%). Notably, the biomarker panel afforded an AUC of 0.844 (95% confidence interval [CI]: 0.806–0.880) toward early ESCC diagnosis. This work highlighted the potential of metabolic analysis for accurate screening and early detection of ESCC and offered insights into the metabolic characterization of diseases including but not limited to ESCC.