Mycotoxins, toxic secondary metabolites produced by fungi, pose a significant threat to food safety and human health. The occurrence of mycotoxins in food commodities necessitates accurate and reliable detection methods. Advanced detection techniques, such as chromatographic techniques and immunochemical assays, have improved sensitivity and specificity. However, the lack of standardized reference material, particularly in less privileged countries, hinders method validation and proficiency testing, ultimately affecting mycotoxin testing and regulation. Moreover, these techniques are complex as they require specialized equipment, and well-trained personnel, thus limiting their practical applications. This comprehensive review provides an up-to-date overview of the occurrence of mycotoxins and recent advancements in detection methods. It examines the crucial role of mycotoxin standards as reference materials for ensuring reliable results in mycotoxins analysis in agriculture commodities. The review addresses emerging challenges, knowledge gaps, and future research directions in mycotoxin detection and reference material development. By synthesizing existing literature, this review aims to provide valuable resources for researchers, policymakers, and other stakeholders in food safety, highlighting the importance of integrated approaches to mitigate mycotoxin contamination and ensuring food safety.