In the past few decades, protein-templated fluorescent gold nanoclusters have attracted much attention in the field of biosensing as their excellent optical properties and good biocompatibility. In this work, a fluorescence turn-on biosensor was fabricated for the sensitive detection of 5-hydroxytryptamine (5-HT) with transferrin encapsulated gold nanoclusters (Tf-Au NCs). The Tf-Au NCs were facilely prepared with the assistance of microwave radiation in several minutes and exhibited good monodispersity as well as strong red-emission. It was demonstrated that an aggregation-enhanced emission of Tf-Au NCs was triggered by 5-HT due to the high affinity between 5-HT and the sialic acid (SA) residues of transferrin. Based on these findings, a highly selective turn-on fluorescent sensor was fabricated for the quantitative determination of 5-HT in the range of 0.2–50 μM (R2 = 0.994) with a detection limit of 0.049 μM (S/N = 3). The developed sensor was also successfully applied for the 5-HT detection in human serum with satisfactory recoveries of 96.20–108.6%.