In this study, ultrasound coupled with subcritical water (USW) technology was employed to extract S. platensis protein, and its inhibitory effect on three enzymes (alpha-amylase, alpha-glucosidase and dipeptidyl peptidase-4 (DPP-IV)) was evaluated. Subsequently, using insulin resistant-HepG2 cell model, the data presented that the USW-extracted protein significantly (p < 0.01) increased glycogen content, hexokinase and pyruvate kinase activities. Then, 11 peptides were identified from intact protein or the HPLC-separated protein fractions by LC-MS/MS. After in silico prediction, 3 peptides (GVPMPNK, RNPFVFAPTLLTVAAR and LRSELAAWSR) were synthesized, and their alpha-amylase, alpha-glucosidase and DPP-IV inhibition activities were validated. Among them, LRSE-LAAWSR displayed the medium activity on alpha-amylase (IC50 = 313.6 mu g/mL), but the best activities on alpha-glucosidase (IC50 = 134.2 mu g/mL) and DPP-IV (IC50 = 167.3 mu g/mL). In summary, for the first time, anti-diabetes peptides were identified from Spirulina platensis, which are useful for functional food additives and pharmaceuticals against type 2 diabetes.