Abstract In the paper, a simple and sensitive electrochemiluminescence (ECL) DNA sensor based on graphene-modified porous Au-paper working electrode (GR/Au-PWE) and calcium carbonate/carboxymethyl chitosan (CaCO3/CMC) hybrid microspheres @ luminescent silver nanoparticles (AgNPs) composites was developed. The GR/Au-PWE with excellent conductivity was successfully prepared for the immobilization of capture probe. The CaCO3/CMC hybrid microspheres were prepared by the precipitation of calcium carbonate in an aqueous solution containing CMC. The AgNPs was synthesized by thermal reduction of silver ions in glycine matrix, taking advantage of the solid-state matrix to control the nucleation and migration of reduced silver atoms. The [email protected] composites exhibited 3.6 times higher ECL intensity than the pure AgNPs-labeled reporter DNA. Taking advantage of dual-amplification effects, the paper-based DNA sensor could detect the target DNA quantitatively, in the range of 4.0×10−17–5.0×10−11M, with a limit of detection as low as 8.5×10−18M, and perform excellent selectivity. The simple, low-cost, sensitive device could be easily applied for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries.