In this study, polymer-supported graphene-based material was designed for electrochemical determination based on the hybridization of DNA. First, dispersions containing graphene oxide (GO) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) polymer in different compositions were transformed into a hydrogel form through the hydrothermal treatment, then 3D and flexible PEDOT:PSS/reduced graphene oxide (RGO) composite sponge material was prepared by using a freeze-drying process. Structural and morphological characterizations showed that PEDOT:PSS polymer served as a skeleton by covering the graphene layers like a mesh and provided an increment in both mechanical strength and electrochemical performance by relatively reducing pore size in 3D material. PEDOT:PSS/RGO composite sponge has high conductivity (158 S cm(-1)) for utilization as an electrode and durability to lift 700 times its weight. For electrochemical determination based on the hybridization of DNA on PEDOT:PSS/RGO composite sponge material, single-stranded DNA (ssDNA) and methylene blue (MB) marked ssDNA probes were immobilized on PEDOT:PSS/RGO composite sponge material at optimum conditions. This sensor achieved a detection limit down to 17 fM with the linear range of 50 fM to 2 mu M for the determination of hybridized DNA. By possessing flexible, free-standing, and durable features, PEDOT:PSS/RGO composite sponge appeared to be a promising electrode material for detection studies.