SP1 (Specificity Protein 1), a member of Sp/KLF protein family, is one of the earliest identified transcription factors, participates in the transcriptional regulation of many genes. 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 exhibited high preference for the codons of CUG and AUC. The mean of effective number of codon (ENC) of SP1 was 50.57 and the value of 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 is little difference between the phylogenetic analysis based on the CDS sequences of SP1 and the clustering analysis based on RSCU. E.coli was the most suitable heterologous expression system for grass carp SP1. The study has shown that CUG and AUC are the optimal codons of SP1 in 4 cyprinids, there are differences in codon preference between species, and natural selection is the main influencing factor leading to SP1 codon preference in 14 species. The model animals zebrafish and mice both could be used as genetic transformation receptors of grass carp SP1. This study provides a theoretical basis for the classification, evolution and expression of SP1 of cyprinidae.