The CRISPR-Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, which has become the focus of molecular diagnosis because it recognizes and cleaves specific DNA or RNA sequences. Using Cas proteins (Cas12, Cas13, Cas14, Cas3, etc.) combined with signal amplification and transformation techniques (fluorescence, potentiometric, colorimetric, lateral flow assay, etc.), researchers have developed many diagnostic platforms with high sensitivity, good specificity, and low cost, which provide a new tool for detecting pathogen nucleic acids. This review presents the biological mechanism and classification of the CRISPR-Cas system, and also summarizes existing technologies for detecting pathogenic nucleic acids based on the trans-cleavage activity of Cas proteins, commenting their properties, functions and application scenarios, with future applications prospected based on the functional characteristics of the CRISPR-Cas system, which is expected to become an ideal detection platform for other multiple targets.