Theaflavins from black tea effectively improve insulin secretion in obesity and diabetes, but the molecular mechanisms are unclear. Here, the palmitic acid (PA)-induced pancreatic β-TC-6 cells and high fat-/high glucose-induced zebrafish were used. The effects of theaflavin-3,3′-digallate (TF3) on glucolipotoxicity-induced insulin secretion dysfunction, ferroptosis and endoplasmic reticulum (ER) stress were investigated by a variety of molecular biological approaches, inductively coupled plasma‒mass spectrometry, transmission electron microscopy and widely targeted metabolomics analysis. TF3 was found to potently inhibit glucolipotoxicity-induced insulin secretion dysfunction and ferroptosis in β-TC-6 cells and zebrafish, with increasing glutathione peroxidase 4 (GPX4) expression, suppressing lipid peroxidation and iron accumulation and protecting mitochondria. Additionally, TF3 attenuated ER stress by regulating three unfolded protein response (UPR) pathways in β-TC-6 cells, and significantly modulated linoleic acid metabolism and L-kynurenine (Kyn) signalling in zebrafish. The expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) was obviously enhanced by TF3. Thapsigargin (Tg), a SERCA2 inhibitor, remarkably reversed the effects of TF3 on insulin production, ferroptosis, ER stress and the Kyn signalling. Together, this work revealed for the first time the critical role of SERCA2 in ferroptosis regulation, and demonstrated TF3 targeted SERCA2 to inhibit ER stress and ferroptosis, thereby protecting β-cell secretory function from glucolipotoxicity.