Abstract: To comprehend the codon usage bias of chloroplast genome in Malania oleifera, the 33 coding DNA sequences were analyzed to acquire the results of neutrality plot, ENC−plot, PR2−plot analysis and determine the optimal codons using software Codon W 1.4.2 and CUSP. The results showed that the GC content at third position of codon was 28.43%, which was much less than that at the first position of codon (47.74%) and at second position (41.05%), there were 30 codons when RSCU value > 1, and the ending of 12 codons were A and 16 codons were U, suggesting that the third position of codons were rich in A and U. There was no significant correlation between GC₁₂ and GC₃, the correlation coefficient was 0.1646, and the regression coefficient was 0.0004; the range of ENC(effective number of codon) value was from 40.39 to 54.86, there were 25 genes when the ENC value was higher than 45, and the ENC ration was mainly distributed beyond −0.05–0.15. Most chloroplast genes of M. oleifera distributed in the lower half or the lower right half of PR2−flat, suggesting that the codon usage bias of chloroplast genome of M. oleifera was more likely to be affected by selection, but also affected by other factors, simultaneously. The optimal codon of chloroplast genome in M. oleifera was identified as UUU, UUA and GUA and other 15 codons.