<jats:title>Abstract</jats:title><jats:p>The 14‐3‐3 family of proteins is central to the regulation of signaling pathways driven by serine/threonine kinases. In humans, 14‐3‐3 consists of seven highly conserved isoforms, yet the function of each isoform remains to be fully elucidated. Synthetic agents capable of isoform‐specific fluorescent labeling of 14‐3‐3 would provide a useful tool for studying in depth the biological roles of isoforms. In this study, the 14‐3‐3σ isoform was evaluated, which possesses a unique Cys38, and a natural product‐based fluorescent labeling agent was designed by introducing an acrylamide group and a fluorescent dye to fusicoccin (FC). In vitro evaluation demonstrated that 12‐hydroxy <jats:bold>1</jats:bold> and <jats:bold>2</jats:bold> exhibit 14‐3‐3σ selective labeling activity over 14‐3‐3<jats:italic>ζ</jats:italic> in the presence of a mode‐3 phospholigand. Furthermore, <jats:bold>2</jats:bold> was shown to label 14‐3‐3σ in cell lysate in the presence of a C‐terminal mode‐3 phosphopeptide derived from ER<jats:italic>α</jats:italic>, with no apparent nonspecific labeling. These results indicate that <jats:bold>2</jats:bold> is capable of selective fluorescent detection of 14‐3‐3σ upon binding to mode‐3 phospholigand under biologically relevant conditions.</jats:p>