As a byproduct of blueberry fruit harvesting, blueberry leaves contain natural compounds with excellent antioxidant activity, presenting significant development potential. This study aims to evaluate antioxidant capacity of blueberry leaves, analyze the bioactive compounds using High-performance Liquid Chromatography (HPLC), and explore their application in the green synthesis of gold nanoparticles (AuNPs). First different solvents—water, ethanol, and methanol—were employed to extract blueberry leaves, and the water extract showed the highest antioxidant activity through ABTS and DPPH assays, along with the Folin–Ciocalteu method for determining total phenolic content. Among all extracts, the water extract showed the highest antioxidant activity and phenolic content. Further analysis by HPLC revealed that the water extract contained a significant amount of Chlorogenic acid (CGA), a common plant-derived phenolic acid with strong antioxidant and reducing properties. Based on these results, the water extract was employed as a natural reducing agent to replace sodium citrate for the green synthesis of AuNPs. The formation and morphology of AuNPs were characterized using UV–visible spectroscopy (UV–Vis) and Transmission Electron Microscopy (TEM). AuNPs synthesized using biomaterials possess the potential to exhibit particle characteristics comparable to those produced by conventional methods, enabling their further application in biomedical researches.