In this study, by injecting cold plasma into the silk fibroin (SF) solution, and adding poly (3,4-ethylenedioxythiophene): polystyrene sulfonic acid (PED-OT:PSS) into the SF solution, the conductive SF-PEDOT:PSS hydrogel modified by plasma was successfully prepared. The changes of the electrochemical parameters and structure of SF-PEDOT:PSS hydrogels after plasma modification were observed with plasma discharge time (60, 120, 180 s) and the type of working gas (Ar, air, He) as variables. The results of electrochemical impedance spectroscopy show that both argon plasma and helium plasma can reduce the impedance of SF-PEDOT:PSS hydrogel in the extremely low-frequency range (10 −2 -10°Hz), and a longer discharge time shrinks the impedance, while the impact of air plasma on the impedance of hydrogel is not obvious. The capacitance results show that both argon and helium plasma can make the capacitance of hydrogel larger, and the longer discharge time leads to the larger capacitance. While air plasma can reduce the capacitance of hydrogel. The results of Fourier infrared absorption spectroscopy and X-ray diffraction spectrum show that the secondary structure of SF is mainly β-fold. The results of Raman show that PEDOT:PSS is successfully incorporated into hydrogel, and there exists random coil conformation of SF in hydrogel. The results of X-ray photoelectron spectroscopy show that the SF-PEDOT:PSS hydrogel mainly contains C, O, and N elements.