A robust, selective and highly sensitive chemiluminescent （CL） platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification （RCA） as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence. This aptamer-primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin-horseradish peroxidase （SA-HRP） were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique .will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity.