Surface plasmon-enhanced electrochemiluminescence (SPEECL) with excellent sensitivity and simplicity has attracted increasing attention. In this work, we reported a novel SPEECL with DNA templated silver nanoclusters (DNA-AgNCs) as ECL emitters and gold nanoparticles (AuNPs) as localized surface plasmon resonance (LSPR) source. The SPEECL with DNA-AgNCs as ECL luminophores possessed low toxicity and avoided the labeling process, which is favorable for its further sensing application. In addition, by investigation of the SPEECL under different distances between DNA-AgNCs and AuNPs, it was demonstrated that the SPEECL was distance dependent. Meanwhile, the SPEECL intensity changed with the sizes and interdistance of AuNPs under different electrodeposition time. Furthermore, by the combination of a cyclic amplification process with enzyme-free catalytic hairpin DNA, a sensitive SPEECL biosensor was proposed for the detection of microRNA (miRNA-21) successfully with a wide linear range from 1 aM to 10 fM and a relatively low detection limit of 0.96 aM, which was applied in the detection of miRNA-21 in real samples with satisfying results. This novel, simple, sensitive, and selective SPEECL with label-free and low-toxic ECL emitters displayed a great potential for bioassay application.