The sea cucumber Apostichopus japonicus was valuable resource for nature and food. High temperature and hypoxia were main kinds of stressors for A. japonicus. In the present study, the global ubiquitinome intestine tissue from four groups (Control, HT, LO and HT_LO) were obtained by label-free quantitative method (LFQ) and liquid chromatography with tandem mass spectrometry analysis (LC-MS/MS). The statistical analysis of quantification values in ubiquitination sites revealed that a total of 523, 387 and 804 genes were differentially expressed in HT, LO and HT_LO in comparison with Control group (P-value ≤ 0.05). Our research may further help reveal the mechanism of stress response from the perspective of protein ubiquitination.