Bioactive natural products (BNPs) from medicinal plants are one of the most successful sources for new drugs. As BNPs contain the beneficial and toxic substances, quantitative analysis of BNPs could not only guarantee the quality of beneficial substances, but also ensure the safety of toxic substances in biological samples, traditional Chinese medicines as well as related products. Although BNPs analysis remains a bottleneck to obtaining the desired performance owing to the trace concentrations and complex sample matrix, much progress has been achieved in the development of detection techniques in recent years. Among them, immunoassay based on specific antibody as the recognition elements has been regards as the most promising candidate for rapid monitoring of BNPs, because of its rapid response, high sensitivity, and low cost. Compared with traditional polyclonal and monoclonal antibody, single-chain variable fragment (scFv) has several desirable characteristics, which has been recognized as a promising alternative in immunoassay. Until now, the obtained scFv has been extensively utilized in the field of environmental monitoring, food safety, and medical diagnosis; however, the application of scFv for BNPs detection must be strengthened. Therefore, this review initially summaries the numerous efforts to produce specific scFv against BNPs. Then, the recent advances of various scFv-based immunoassays for BNPs analysis are particularly emphasized. Meanwhile, the real-world application of these isolated scFvs for BNPs detection, along with their advantages, potential limitations, current challenges as well as future trends, are systematically discussed. It hopes to provide rewarding insights to the future development and board application of novel scFv-based immunoassay for BNPs monitoring in different fields.