Abstract: 【Objective】To analyze codon usage bias of the mitochondrial genome of Charonia tritonis and to explore effects of gene mutation and natural selection on codon usage bias，so as to provide reference for phylogenetic tree construction，germplasm resources protection and genetic improvement of C. tritonis.【Method】Mitochondrial genome of C. tritonis was taken as material and 11 CDSs（Coding DNA sequences）from the whole genome sequence of C. tritonis mitocondria were screened out，which were non-repeated sequences more than 300 bp and started from ATG，by using the software of CodonW 1.4.2 for codon usage bias analysis and optimal codons were selected with △RSCU>0.08 and RSCU> 1.00 in highly expressed gene bank.【Result】GC content in position 1（GC1）ranged from 35.80% to 53.10% with an average of 43.54%. GC content in position 2（GC2）ranged from 32.40% to 43.40% with an average of 36.11%. GC content in position 3（GC3）ranged from 23.00% to 30.60% with an average of 27.79%. GC1 and GC2 contents were obviously higher than GC3 content. GC content in position 3 of synonymous codon（GC3s）ranged from 20.90% to 29.60% with an average of 26.23%，suggesting that codon third-base mainly ended with A/U（T）. Codon adaptation index（CAI）ranged from 0.096 to 0.166 with an average of 0.137. Codon bias index（CBI）ranged from -0.212 to -0.086 with an average of -0.133. Frequency of optimal codons（FOP）ranged from 0.246 to 0.339 with an average of 0.306. Effective number of codon（ENC）ranged from 44.37 to 51.69 with an average of 47.84. Grand average of hydropathicity（GRAVY）ranged from 0.4419 to 1.4727 with an average of 0.9501. Neutrality plots，ENC-plot and correspondence analysis results showed that the codon bias of mitochondrial genome in C. tritonis was affected by both gene mutation and natural selection but dominantly by the latter. Finally，considering high frequency codons and high expression codons，11 codons were identified as the optimal codons in the mitochondrial genome of C. tritonis，and they were UUU，CUA，GUA，UAU，CAA， AAA，UCA，CCA，ACA，GCC and GCA，among which one ended with G/C，and the rest 10 ended with A/U.【Conclusion】The results show that the codons prefer to end with A/U（T）base，and the 11 screened optimal codons prefer to end with A/U base in mitochondrial genome of C. tritonis. In mitochondrial genome of C. tritonis，the codons show weak bias，natural selection play a leading role in the formation of codon usage bias.