Abstract: EMBOSS and Codon W software were used in order to analyze the usage preference of S-RNase gene codon in Lycium barbarum. The codon preference of S-RNase gene was compared with that of 10 species such as yeast, Escherichia coli and citrus. The results showed that the S-RNase codon preference ended with A/T; RSCU value showed that there were 9 codons used in high frequency of this gene, and there was obvious codon preference difference compared with other species of homologous genes. Clustering results based on S-RNase gene coding sequences more accurately reflected the affinity between species than the use of preferred analysis results. Compared with other species, the codon usage frequency of L. barbarum S-RNase gene was smaller in the same family and larger in different families. At the same time, S-RNase gene of L. barbarum was more suitable for the heterologous expression in yeast by comparing with the usage frequency, but it needed to be optimized and modified properly. Preference of S-RNase codon in L. barbarum laid a theoretical foundation for heterologous expression and genetic research.