Hypoxia plays an important role in regulating a variety of physiological responses as well as in pathological situations, but to date the roles of Toll-like receptors (TLRs) in fish in response to hypoxia are still poorly understood. Here, we sequenced the transcriptome of G. eckloni and identified the members of TLR family by scanning transcriptome, and then investigated the expression profiles of a complete set of TLRs in G. eckloni in response to acute hypoxia (4 h at DO = 0.3 ± 0.1 mg/L). The de novo-assembled transcriptome consisted of a total of 162,235 transcripts, further clustered into 110,231 unigenes. Based on the transcriptome, a total of 18 TLRs were identified in G. eckloni, and of them three TLRs (TLR5, TLR8 and TLR22) possessed two distinct paralogous genes. The duplicated genes of TLR22 were discovered for the first time in cyprinid fish, but did not origin from a recent duplication event. Of them TLR22b may be specific for schizothoracine fish, at least for G. eckloni. Phylogenetic analysis supported the classification of TLRs into six families as in other vertebrates but was partly different from the previous study. The sliding window analysis showed strong signals of positive selection in TLR2, TLR 4, TLR 5a, TLR 7, TLR 19, TLR 20, TLR 21, TLR 22a and TLR 22b, but most codons under positive selection were located in the putative LRR regions. The mRNA expression of most TLRs in head kidney, spleen and gill decreased significantly or remained unchanged under acute hypoxia, whereas acute hypoxia increased expressions of TLR2 and TLR3 in head kidney, of TLR8a, TLR12 and TLR19 in spleen, and of TLR1 in gill, suggesting tissues-specific expressions of TLRs play important roles in mediating innate immune responses for host defense against tissue damages or physiological changes induced by hypoxia.