In this work, poly(sodium 4-styrenesulfonate) (PSS)-encapsulated acridinium sulfonamide (AS)-functionalized CoFe 2 O 4 nanoparticles (CoFe 2 O 4 /AS/PSS NPs) were synthesized by a layer-by-layer assembly strategy. Compared with previously reported chemiluminescence (CL)-functionalized magnetic nanomaterials, CoFe 2 O 4 /AS/PSS NPs exhibited a high CL intensity with a much lower synthetic concentration of CL reagent and shorter synthetic time. The reason was found to be attributed to the high loading efficiency of AS molecules, the high CL efficiency of AS, and the catalytic effects of CoFe 2 O 4 NPs and PSS on the CL reaction. Moreover, CoFe 2 O 4 /AS/PSS NPs showed good CL stability and an easy magnetic separation property. On this basis, a CL biosensor for cholesterol detection was constructed by modifying cholesterol oxidase on the surface of CoFe 2 O 4 /AS/PSS NPs using cysteamine-protected gold nanoparticles as a "bridge." It demonstrated a wide linear range of 0.8−200 μM with a detection limit of 0.603 μM, both of which were better than those of most of the reported biosensors. Moreover, the developed CL biosensor required only one incubation step to detect cholesterol, which was simpler than most reported biosensors that require the mixing of nanomaterials with cholesterol oxidase and signal molecules to detect cholesterol. Finally, the detection of cholesterol in human serum samples was performed with good recoveries ranging from 90.5 to 109.6%, demonstrating that the developed CL biosensor is reliable and has great potential for clinical diagnosis. The CL magnetic analytical platform is also promising for application to other hydrogen peroxide-producing systems for the detection of related important biomolecules.