Hypoxia preconditioned plasma (HPP) is generated by cultivating blood under hypoxic conditions, allowing blood cells to naturally regulate the production of growth factors in response to hypoxia, which seems ideal for diabetic wound treatment. This study aims to verify the efficacy and underlying mechanism of HPP on diabetic wound healing. The concentrations of angiogenesis-related factors (VEGF, PDGF, EGF, IGF, TSP1, PF4) measured by ELISA were significantly increased in HPP. In vitro, HPP promoted the proliferation and migration of HUVECs and HSF, and enhanced the tube formation of HUVECs. In vivo, HPP promotes wound angiogenesis and collagen deposition, significantly accelerating diabetic wound healing (84 ± 10% vs. 65 ± 20%, p < 0.01). Furthermore, western blot and qPCR results showed that both in cells and wounds, the expression of pro-angiogenic factors, as well as p-AKT, was increased in HPP. These findings suggest that HPP benefits diabetic wound healing and may represent a promising therapeutic approach for diabetic wound healing.