Abstract: Specificity Protein 1 (SP1), a member of the Sp/KLF protein family, is one of the earliest identified transcription factors and participates in the transcriptional regulation of many genes. This study aimed to explore the codon usage pattern in evolution process and the phylogenetic relationship among different species of SP1 and also provide references for its high heterologous expression. The software such as Codon W, Clustal X, MEGA 4.0, and SPSS were used to analyze the codon bias and evolutionary of SP1 of 4 cyprinidae fish species and 10 other species. The results showed that the Cyprinidae fish SP1 hithly preferred the codons of CUG and AUC. The mean of an effective number of codons (ENC) of SP1 was 50.57 and the value of the codon adaptation index (CAI) was between 0.184 and 0.379, which was far less than 1. The above two indexes illustrated that SP1 in different species showed codon bias. Moreover, the SP1 of four cyprinid fish showed similar codon preference. The ENC-plot analysis revealed that natural selection was the main reason for the SP1 codon usage bias. In cluster analysis, there was little difference between the phylogenetic analysis based on the CDS sequences of SP1 and the clustering analysis based on RSCU. Escherichia coli was the most suitable heterologous expression system for Ctenopharyngodon idella SP1, and the model animals Danio rerio and Mus musculus could both be used as genetic transformation receptors for C. idella SP1. This study showed that CUG and AUC were the optimal codons of SP1 in 4 cyprinids, there were differences in codon preference between species, and natural selection was the main influencing factor leading to SP1 codon preference in the 14 species. This study provides a theoretical basis for the classification, evolution and expression of Cyprinidae SP1.