The precise target recognition and nuclease-mediated effective signal amplification capacities of CRISPR-Cas systems have attracted considerable research interest within the biosensing field. Guided by insights into their structural and biochemical mechanisms, researchers have endeavored to engineer the key biocomponents of CRISPR-Cas systems with stimulus-responsive functionalities. By the incorporation of protein/nucleic acid engineering techniques, a variety of conditional CRISPR-Cas systems whose activities depend on the presence of target triggers have been established for the efficient detection of diverse types of non-nucleic acid analytes. In this review, we summarized recent research progress in engineering Cas proteins, guide RNA, and substrate nucleic acids to possess target analyte-responsive abilities for diverse biosensing applications. Furthermore, we also discussed the challenges and future possibilities of the stimulus-responsive CRISPR-Cas systems in versatile biosensing.