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Advances in materials science has accelerated the development of diagnostic tools with the last decade witnessing the development of enzyme-free sensors, owing to the improved stability, low cost, and simple fabrication of component materials. However, the specificity of non-enzymatic sensors for certain analytes still represents a challenging task, for example, the determination of cholesterol level in blood is vital due to its medical relevance. In this work, a reagent displacement assay for cholesterol sensing in serum samples was developed. It is based on coating of a glassy carbon electrode with a polymer of intrinsic micro-porosity (PIM) that forms a host-guest complex with methylene blue (MB). In the presence of cholesterol, the MB electroactive probe was displaced due to the stronger association of cholesterol guest to the PIM host. The decrease in the oxidative current was proportional to the cholesterol concentration achieving a detection limit of approximately 0.1 nM.