The sea cucumber Holothuria leucospilota is an edible sea cucumber species, which is widely distributed in the shallow waters of tropical and subtropical Indo-Pacific Ocean. Nowdays, H. leucospilota has become an important fishery resource owing to the continuously increasing market demand, however, overfishing activities will put this species at risk of extinction in the future. Artificial breeding of H. leucospilota is considered as a useful strategy to solve this problem. The use of suitable substitute diets is an economical and efficient approach for breeding of planktonic larvae of sea cucumbers. In this study, the microalgae Chaetoceros muelleri and Rhodotorula sp. were fed to planktonic larvae of sea cucumber H. leucospilota, either singly or in combination, to evaluate their nutritional quality. C. muelleri and Rhodotorula sp. were mixed in 4∶0, 3∶1, 2∶2, 1∶3 and 0∶4, by volume, and they were labeled as groups A, B, C, D and E, respectively. Results showed that larvae in group A, which fed solely on C. muelleri, exhibited the fastest growth and development among the five groups, larvae could develop to the auricularia stage on the 10th day and the doliolaria stage on the 18th day; meanwhile, survival of the larvae in group A was the highest, and the survival rate in this group was 58.73%±2.75% at the end of the experiment. By contrast, the larvae in groups D and E always stayed in the early auricularia stage, and individuals in these two groups could not form the hyaline spheres during the whole study period. The larvae in group E exhibited negative growth during the whole study period, and survival rate of this group was only 22.22% ±5.50% at the end of the experiment. In general, negative effects on the development, growth and survival of the planktonic larvae of H. leucospilota were found with the increase in the proportion of the Rhodotorula sp. in the diet, and therefore Rhodotorula sp. was not a suitable substitute for breeding of H. leucospilota.