Actinomycetes are an important source of natural antibiotics, and have a wide variety of transcription factors that precisely control the biosynthesis of antibiotics as secondary metabolites. As an important family of prokaryotic single-component signaling systems, TetR family transcriptional regulators (TFRs) are involved in the regulation of multiple cellular activities such as antibiotic biosynthesis, drug efflux, primary metabolism. On the basis of the recent studies in our lab and the research advances published, we review here the molecular regulatory mechanism of TFRs involved in the biosynthesis of several important antibiotics from the perspective of regulated target genes, and outlined the ligands of TFRs. Finally, this review summarizes and indicates the application of TFRs in increasing antibiotic production, activating silent cryptic gene clusters and artificially exploiting synthetic biology elements.