A novel electrochemical sensing approach was developed to detect DNA, RNA, and protein targets, utilizing the host–guest interaction between methylene blue (MB)-labeled probe and β-cyclodextrin-based nanocomposite (rGO/β-CDP). During the assay, the MB-labeled probe interacted with the corresponding target to form a probe/target complex. Due to steric hindrance, the formation of this complex reduced the amount of MB-labeled probes that can be captured by the β-cyclodextrin-based nanocomposite on the electrode surface, resulting in a measurable decrease in the electrochemical signal. Upon optimization, the sensing platform exhibited satisfactory analytical performance for three cancer-related model analytes: p53 DNA, microRNA-21 RNA, and thrombin protein, achieving low detection limits of 3.4 nM, 4.4 nM, and 5.7 pM, respectively. In this work, the electrochemical assay separates the detection process into two stages: target recognition in solution and signal transduction on the electrode surface. This fabrication effectively circumvents the issues associated with nonspecific conformational changes and the complicated probe installation procedures on electrode surfaces that are common in traditional sensing platforms. Integrating host–guest recognition with electrochemical assay, the developed approach not only advances the understanding of host–guest chemistry in sensor applications but also establishes a foundation for future innovations in the detection of biomarkers.