Peroxidases are essential enzymes that catalyze redox reactions, with wide-ranging biological implications. Among these, an enhanced ascorbate peroxidase (APEX) has emerged as a valuable tool for studying intricate intracellular events with spatiotemporal precision, particularly in protein–protein, protein–RNA, and protein–DNA interaction networks in living cells. This review discusses APEX’s structural and functional attributes, its evolution through genetic engineering, and its transformative applications in high-resolution mapping used for proteomic and transcriptomic studies. Furthermore, it highlights recent advancements in substrate innovation and addresses current challenges and future directions in leveraging APEX for cutting-edge biological research.