Timely diagnosis is of great benefit to improve the survival rate of cancer patients. Body fluid cancer biomarker detection is a critical kind of noninvasive method for cancer diagnosis. Nevertheless, traditional methods for cancer biomarker detection always rely on a large-scale instrument and involve sophisticated operation. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, holding great promise as the next-generation molecular diagnostic technology. In this Feature, we introduce the working mechanisms of different kinds of CRISPR/Cas systems for biosensing and CRISPR/Cas-mediated detection strategies for different kinds of cancer biomarkers including nucleic acids, proteins, and extracellular vesicles. In addition, the perspective and challenges of CRISPR/Cas-based strategies for cancer biomarkers are discussed.