Abstract: The analysis of the identity, the codon usage frequency and the codon usage preference were made to sequences of Carassius auratus auratus serum transferrin (Tf) cDNA cloned in our laboratory and 14 fish Tf cDNA from reference documentation by DNAman v4.0. The results show that the identity of Tf cDNA sequences among Cypriniformes fishes is more than 46% and the identity of Tf cDNA sequences among Oncorhynchus fishes is between 34%~99% with larger variation. The higher the codon usage frequency or the lower the codon usage preference, the more important the effect of amino acids coded by the codon to form protein space structure and perform basic physiological functions. Furthermore, the earlier the species differentiation time on evolution, the lower the difference of the codon usage frequency and preference and the higher by contraries. Moreover, species with near homology relationship on evolution share the near codon usage frequency and preference index or the similar index. According to the codon usage frequency and the codon usage preference analysis, the evolution sequence of Tf gene, viz. Cypriniformes, Melanogrammus, Oncorhynchus and Paralichthys, were presumed.