<p>CRISPR/Cas systems have revolutionized nucleic acid recognition, offering unparalleled programmability for biosensing. Simultaneously, DNA nanomachines have emerged as powerful bioanalytical tools due to their structural precision, biocompatibility, and multifunctional capabilities. The strategic integration of these platforms synergistically enhances sensitivity, specificity, and multiplexing potential, creating next-generation bioanalytical systems. While promising, this convergence presents unique engineering challenges and necessitates critical evaluation. This review systematically analyzes the target recognition mechanisms of CRISPR/Cas systems and critically evaluates prevalent signal readout modalities. A core focus is the critical assessment of innovative biosensing strategies that leverage DNA nanomachines—particularly dynamic walker systems—coupled with CRISPR/Cas activation. We highlight representative integrated platforms, detailing their operational principles and objectively examining their demonstrated advantages against inherent limitations such as signal leakage, complex assembly, and in vivo applicability constraints. By providing a critical analysis of the synergistic potential and current constraints of CRISPR/Cas-DNA nanomachine integration, this review aims to guide rational design towards robust, clinically translatable precision diagnostics and therapeutics.</p> Graphical abstract <p>Strategies for coupling CRISPR/Cas systems with DNA nanomachines in biosensing</p> <p></p>