Abstract For high recoveries and purities of L-proline (L-Pro) and L-hydroxyproline (L-Hyp) from the low-value sea cucumber Acaudina leucoprocta, the extraction process was systematically investigated and optimized. The results indicated that, in decoloration of A. leucoprocta hydrolysate, activated carbon powdered W660 was more suitable due to the low addition amount and short treatment time compared with macroporous resin HZ816 offering comparable decoloration ratio and imino acids recovery yields. For oxidative deamination, HCl-HNO3 exhibited better performance than NaNO2 with complete primary amino acids broken-down and fewer imino acids loss. After decoloration and oxidative deamination under their optimized conditions, an extraction solution was obtained with L-Pro and L-Hyp recovery yields of 76.3% and 78.8% from A. leucoprocta hydrolysate, which was directly used for separation of L-Pro and L-Hyp with ion-exchange chromatograph. This work may be referenced for the large-scale L-Pro and L-Hyp production from other low-value materials.