Nontoxic substitutes to mycotoxins can facilitate the development of eco-friendly immunoassays. To explore a novel nontoxic substitute to ochratoxin A (OTA), this study screened shark anti-idiotypic variable new antigen receptors (VNARs) against the alpaca anti-OTA nanobody Nb28 through phage display. After four rounds of biopanning of a naïve VNAR phage display library derived from six adult Chiloscyllium plagiosum sharks, one positive clone, namely, P-3, was validated through a phage enzyme–linked immunosorbent assay (phage ELISA). The recombinant anti-idiotypic VNAR AId-V3 was obtained by prokaryotic expression, and the interactions between Nb28 and AId-V3 were investigated via computer-assisted simulation. The affinity of AId-V3 for Nb28 and its heptamer Nb28-C4bpα was measured using Biacore assay. Combining Nb28-C4bpα with AId-V3, a novel direct competitive ELISA (dcELISA) was developed for OTA analysis, with a limit of detection of 0.44ng/mL and a linear range of 1.77-32.25ng/mL. The good selectivity, reliability, and precision of dcELISA were confirmed via cross-reaction analysis and recovery experiments. Seven commercial pepper powder samples were tested using dcELISA and validated using high-performance liquid chromatography. Overall, the shark anti-idiotypic VNAR was demonstrated as a promising nontoxic substitute to OTA, and the proposed method was confirmed as a reliable tool for detecting OTA in food. Environmental ImplicationOchratoxin A (OTA) is a common mycotoxin in food and has been listed as a Group 2B human carcinogen by IARC. In traditional immunoassays, chemical synthesis of OTA artificial antigens requires abundant OTA standard and organic solvent, which threatens the operator’s health and environmental safety. This manuscript reports a novel nontoxic substitution to OTA (anti-idiotypic VNAR) that was screened with anti-OTA alpaca nanobody from the naïve phage display shark VNAR library. The nanobody and anti-idiotypic VNAR were then used to develop a novel direct competitive ELISA to detect OTA in pepper, which is economical and eco-friendly.