As a crucial biomarker for some diseases, the determination of cholesterol in human serum is of great significance for the diagnosis and prevention of these diseases. Hence, a portable cholesterol detection method is necessary for clinical and domestic applications. Here, a portable paper sensor was designed for cholesterol detection by modifying screen-printed electrode (SPE) with nanoporous gold (NPG). To achieve the reliable cholesterol detection, a synergistic strategy was proposed based on the oxidation of cholesterol by cholesterol oxidase (ChOx) and the reduction of oxidation product (HO) by NPG. Compared to existing electrochemical sensors, the resulting paper sensor exhibited a wider linear response in a range from 50 μM to 6 mM as well as a higher sensitivity of 32.68 μA mM cm with a lower detection limit of 8.36 μM. Moreover, the portable paper sensor presented strong anti-interference capability and stability in the detection of cholesterol in human serum, and the data detected by the portable paper sensor were consistent with that obtained by an automatic biochemical analyzer. These unique performances confirmed that the proposed paper sensor was a sensitive, reliable, and portable cholesterol detection method, making it a good choice for cholesterol detection.