Recurring mycotoxins contamination has posedaseriousthreatto food safety worldwide. Competitive immunoassays are widely used techniques for high-throughput mycotoxins detection in agricultural products and foods. However, the inevitable introduction of mycotoxin conjugates produced by chemical conjugation usually results in complicated by-products, large batch errors and threats to operators and environment. Biologically derived surrogates of mycotoxin conjugates or mycotoxin standards are renewable immunoreagents. They can serve the same function as the responding counterparts in the immunoassays. The substitute-based immunoassays exhibit satisfactory sensitivity, pose less health threats to operators and environment, and contribute to the standardization of immunoassays for mycotoxins. This review focuses on the current applications of substitute-based immunoassays, clarifies their underlying mechanisms and provides a careful comparison. Challenges and future prospects are discussed.