Surface-enhanced Raman scattering (SERS) is a non-invasive spectroscopic technique that provides specific chemical fingerprint information for biomarkers in cancer and pathogen diagnosis. However, the SERS strategies are limited by the non-specific interactions between substrates and co-existing substances in biological matrices and the challenges of obtaining molecular fingerprint information from the complex vibrational spectrum. In recent years, the rapid development of novel substrates with high SERS activity has opened up new opportunities for their applications in cancer and pathogen diagnosis. The aim of this review is to present the recent progress and perspectives of novel SERS-based substrates for cancer and pathogen diagnostic applications. First, we will introduce recently developed SERS-active nanomaterials and discuss the influencing factors of the SERS signals. Second, the advantages of SERS in the diagnosis of cancer and pathogens will be given. Third, we will review the latest breakthroughs in cancer and pathogen detection research with SERS technology, as well as the new opportunities for SERS applications brought about by artificial intelligence (AI) technology. In addition, the novel microfluidic-SERS platforms for cancer and pathogens diagnosis will also be discussed. Finally, we will summarize the challenges and future perspectives of SERS technology in the field of early cancer diagnosis and rapid pathogen detection. It is highly expected that this review could benefit a comprehensive understanding of the research status of the SERS-active nanomaterials and arouse the research enthusiasm for them, leading to accelerated clinical translation of SERS technology in cancer and pathogen diagnosis.