A novel and enzyme-free electrochemical sensor was designed for multiplexed DNA detection based on a surface confined DNA assembly enabled target recycling amplification. In this designed system, two hairpin DNA probes (hairpin 1 and hairpin 2) were immobilized on the surface of gold nanoparticles/graphene/nafion modified glassy carbon electrode (AuNPs/Gr/Nf/GCE). In the presence of two DNA targets, the hairpin structures of hairpin 1 and hairpin 2 opened through the complementary hybridization, resulting in the formation of hairpin 1: target DNA 1 and hairpin 2: target DNA 2. When they met methylene blue (MB)-labeled hairpin 3 (hairpin 3-MB) and ferrocene (Fc)-labeled hairpin 4 (hairpin 4-Fc), DNA cyclic amplification was activated, and the DNA targets were released and could be reused for the next recycling. Meanwhile, coupled with the utilization of positively charged gold nanoparticles ((+)AuNPs), multiplexed DNA detection was realized in the concentration range from 10 fM to 1.5 pM with the detection limits of 3.6 fM and 2.5 fM for target DNA 1 and target DNA 2, respectively. Moreover, satisfactory results were obtained when detecting DNA targets in human serum samples, suggesting that this proposed sensing platform is promising for the applications in complex biological samples.