Conditioned medium (CM) is a new treatment modality in regenerative medicine and has shown a successful outcome in wound healing. We recently introduced extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel), an adipose-derived stem cell and adipose native extracellular matrix-enriched product for cytotherapy. This study aimed to evaluate the effect of CM from ECM/SVF-gel (Gel-CM) on wound healing compared with the conventional CM from adipose tissue (Adi-CM) and stem cell (SVF-CM). In vitro wound healing effect of three CMs on keratinocytes and fibroblasts was evaluated in terms of proliferation property, migratory property, and extracellular matrix production. In vivo, two full-thickness wounds were created on the back of each mice. The wounds were randomly divided to receive Gel-CM, Adi-CM, SVF-CM, and PBS injection. Histologic observations and collagen content of wound skin were made. Growth factors concentration in three CMs was further quantified. In vitro, Gel-CM promoted the proliferation and migration of keratinocytes and fibroblasts and enhanced collagen I synthesis in fibroblasts compared to Adi-CM and SVF-CM. In vivo, wound closure was faster, and dermal and epidermal regeneration was improved in the Gel-CM-treated mice compared to that in Adi-CM and SVF-CM-treated mice. Moreover, The growth factors concentration (i.e., vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-β) in Gel-CM were significantly higher than those in Adi-CM and SVF-CM. Gel-CM generated under serum free conditions significantly enhanced wound healing effect compared to Adi-CM and SVF-CM by accelerating cell proliferation, migration, and production of ECM. This improved trophic effect may be attributed to the higher growth factors concentration in Gel-CM. Gel-CM shows potential as a novel and promising alternative to skin wound healing treatment. But limitations include the safety and immunogenicity studies of Gel-CM still remain to be clearly clarified and more data on mechanism study are needed.