Surface-enhanced Raman spectroscopy (SERS) has evolved from a laboratory technique to a practical tool for ultra-sensitive detection, particularly in the biomedical field, where precise molecular identification is crucial. Despite significant advancements, a gap remains in the literature, as no comprehensive review systematically addresses the high-precision construction of SERS substrates for ultrasensitive biomedical detection. This review fills that gap by exploring recent progress in fabricating high-precision SERS substrates, emphasizing their role in enabling ultrasensitive bio-medical sensors. We carefully examine the key to these advancements is the precision engineering of substrates, including noble metals, semiconductors, carbon-based materials, and two-dimensional materials, which is essential for achieving the high sensitivity required for ultrasensitive detection. Applications in biomedical diagnostics and molecular analysis are highlighted. Finally, we address the challenges in SERS substrate preparation and outline future directions, focusing on improvement strategies, design concepts, and expanding applications for these advanced materials.