Cholesterol is an essential compound for human health, and a high or low concentration of cholesterol is closely related to various diseases. Thus, developing a simple method for POCT of cholesterol has great significance in clinical diagnosis. In this work, alginate (Alg) hydrogels with glow-type chemiluminescence (CL) were prepared and applied for rapid and quantitative cholesterol detection via a smartphone. The glow-type CL hydrogels (HRP/COD/luminol/Alg hydrogels) contained luminol as a chemiluminescent reagent, horseradish peroxidase (HRP) and cholesterol oxidase (COD) for enzymatic cascade reactions. The HRP/COD/luminol/Alg hydrogels exhibited outstanding stability, which effectively avoided the enzyme inactivation during long-term storage. Furthermore, the HRP/COD/luminol/Alg hydrogels exhibited longer and more stable glow-type CL. With the help of COD catalytic specificity for cholesterol and bi-enzymatic cascade reactions, the glow-type CL hydrogels realized the specific and sensitive detection of cholesterol. The smartphone was used as a detector instead of a special large instrument for responding to the glow-type CL emission, and a LOD of 7.2 μM was obtained. Therefore, the proposed sensor expands the application of the glow-type CL in POCT and provides an alternative way for cholesterol detection in clinical diagnosis.