Toll-like receptors (TLRs) are one of the most extensively researched pattern recognition receptors (PRRs) and play an important role in the innate immune system. In this study, partial cDNA sequences of the Pf_TLR18 and Pf_TLR19 genes and complete cDNA sequence of the Pf_TLR21 gene were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORFs) of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes were 1956 bp, 2262 bp and 2949 bp in length, encoding 651, 753 and 982 amino acids, respectively. The Pf_TLR18 and Pf_TLR19 consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor domain, and the Pf_TLR21 only has LRRs and TIR domain. Homologous identity revealed that the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes have high nucleotide and protein sequence similarity with channel catfish, especially the TIR domains that exhibited the greatest conservation compared to channel catfish. Ontogenetic expression analyses indicated that the mRNA expressions of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes could be detected from fertilized eggs to 30 day post-hatching and they exhibited different variation trends after hatching. The three TLR genes were expressed in various tissues, but they were mostly highly expressed in the spleen. The mRNA expression levels of the three genes were up-regulated in the spleen, head kidney, trunk kidney, liver and blood after challenge of killed Aeromonas hydrophila. In addition, the expressions of the three TLR genes were induced to up-regulate in isolated peripheral blood lymphocytes of yellow catfish after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). Our findings indicate that the three TLR genes may play a potential role in the host defense against pathogenic microbes. These results will provide valuable information to better understand the function of TLR genes in the innate immune system of yellow catfish.