The aim of this work was to evaluate the effect of different concentrations of gold nanoparticles (AuNPS) on equine platelet morphology, ultrastructure as well as secretion of bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), gelsolin (GSN), and tumor necrosis factor alpha (TNF-α). The gold nanoparticles, with an average diameter of 20 nm, were incubated with whole anticoagulated blood at 1%, 5%, and 10% w/v for 24 hours. We showed that the application of gold nanoparticles at all the tested concentrations led to platelet activation; however, 5% of AuNPS resulted in the secretion of elevated levels of GSN and VEGF, while the concentration of TNF-α was the lowest. The data indicate that this method may be considered a promising tool for improving the currently used in clinical practice commercially available autologous conditioned serum (ACS) isolation systems for the treatment of equine osteoarthritis.