Abstract: Hemoglobin is one of the most important proteins for aerobic metabolism in vertebrate. The studies of Hb are more in Mammalia, but less in fishes with low-oxygen environment. This study cloned Hba1/2 and Hbb1/2 cDNA sequences of Culter alburnus (C. alburnus) hemoglobin, which encode 143, 143, 147 and 147 of amino acids, respectively. The second structure analysis of proteins indicated that Hba1/2 and Hbb1/2 included 7 and 8 helical regions, 14 and 13 α1β2 interfaces, 12 and 16 heme bindings, 16 and 16 α1β1 interfaces, respectively, and 6 Bohr effect residues only for Hba1/2. Compared with amphibians and mammalia, there were 10 and 5 amino acid substitutions in fishes of Hba and Hbb functional domains, which may be used to adapt to hypoxia. However, compared with tolerant and intolerant hypoxia fishes, we did not find any coherent substitution in the second structure of proteins, indicating that the tolerant hypoxia trait of fish may be regulated by upstream signal pathways. The phylogenetic relationship showed the duplication events of Hba1/2 and Hbb1/2 isoforms may occurre after vertebrate and before teleost of whole-genome duplications. Interestingly, in the phylogenetic trees, the genetic relationships of C. alburnus and Danio rerio of Hba1/2 and Hbb1 were closer than those of C. alburnus and other fishes, probably because both C. alburnus and Danio rerio belong to Cypriniformes and intolerant to hypoxia. This study cloned Hba1/2 and Hbb1/2 cDNA sequences of C. alburnus hemoglobin, analyzed molecular characteristics and phylogenetic relationships with others, and discussed the possible causes of fish tolerance to hypoxia. These findings provide a theoretical basis and potential direction for fish tolerant hypoxia biology.