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A PEG-based hydrogel with a high conductivity and water content was synthesized using a two-step sequential polymerization process by in-situ polymerizing poly(3,4-ethylenedioxythiophene) (PEDOT) within poly(4-styrenesulfonic acid) (PSS)-containing poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix. A small amount of diluted sulfuric acid (H2SO4) was added as an accelerator to increase the conductivity via reduced polymerization time. Among the various molecular weights (MW) of PEG, PEG-DA with MW 3400 was used for first hydrogel due to its mechanical property and water content. Incorporation of PSS within the PEG hydrogel facilitated the in-situ synthesis of PEDOT within the hydrogel, producing a hydrogel with a higher conductivity, which was further enhanced by H2SO4 treatment. The resultant semi-interpenetrating network hydrogel scaffolds were shown to consist of up to more than 80wt% of water with a compressive modulus of 21kPa and an electrical conductivity of 1.69×10−2Scm−1. The surface of the resultant conductive hydrogel could be modified via photochemical fixation for cell adhesion with negligible conductivity change. In vitro studies using electro-responsive H9C2 myocytes showed that the developed hydrogels not only did not exhibit any cytotoxicity but also supported cell adhesion and proliferation. This work demonstrates that the architectural design of the conductive hydrogel scaffolds and growth mechanism of PEDOT in the hydrogel platform play a pivotal role in determining the properties of the resulting conductive hydrogel. The attractive performance of these hybrid hydrogels will open a new approach for the further research on electrically conductive tissue engineering scaffolds.