In recent years, gold nanoparticles (AuNPs) have attracted much attention due to their extensive applications in fields such as biomedicine, electronics, catalysis, and environmental science. However, traditional chemical methods for AuNPs synthesis present certain challenges, such as the use of harsh chemicals and high energy consumption. These limitations have led to the development of alternative, sustainable synthesis methods that are efficient, cost-effective, and environmentally friendly. These methods focus on the principle of green chemistry, utilizing renewable biomass sources (e.g., plant tissues, bacteria, fungi, and algae) and nontoxic solvents to minimize environmental impact. Biomolecules derived from biomass, such as polyphenols, proteins, and unsaturated fatty acids, enable the synthesis of AuNPs under mild and eco-friendly conditions. This review provides a comprehensive overview of recent advancements in the sustainable synthesis and applications of AuNPs. It summarizes the specific active compounds that drive the reduction and stabilization of AuNPs. It also explores the characterization techniques and underlying mechanisms involved in synthesis. Furthermore, their cellular effects and long-term safety are discussed, along with their extensive applications in biomedical fields, including bioimaging and cancer therapies. Finally, the potential of AuNPs is summarized, highlighting future perspectives as well as emerging opportunities and challenges in biological applications.