In medical and biomedical fields, enzyme-mimetic nanomaterials have garnered significant interest as efficacious signal enhancers for biocatalyst-linked immunosorbent assays (BLISA). Despite the burgeoning enthusiasm, engineering artificial biocatalysts that exhibit both exceptional catalytic proficiency and pronounced colorimetric signal output remains a formidable challenge. Inspired by the heme structures and biocatalytic activities of horseradish peroxidase, we introduce the synthesis of vanadium single-atoms (SAV) coordinated artificial peroxidases as BLISA for highly sensitive and selective carcinoembryonic antigen (CEA) immunoassay. Our synthesized SAV exhibits peroxidase (POD)-like activity that is both efficacious and highly specific, surpassing the performance of many other single-atom-structured materials. The SAV-linked immunoassay demonstrates an ultrasensitive response to the target antigen (CEA), with a linear detection range spanning 0.03-10 ng/mL and an impressively low detection limit of 0.335 ng/mL. This straightforward and robust immunoassay technique not only achieves superior signal amplification compared to traditional natural enzymes but also boasts high precision, commendable reproducibility, and remarkable specificity, aligning closely with conventional enzyme-linked immunosorbent assay for CEA detection in serum samples. This study offers a blueprint for designing artificial peroxidase-based colorimetric nanosystems, promoting the evolution of ultrasensitive BLISA applications for the early diagnosis and intervention of cancer.Copyright © 2024 Elsevier Ltd. All rights reserved.