Because the growth activities of cells considerably depend on the surface characteristics of tissue culture substrates, tissue–substrate interface is a crucial factor in modulating the behavior of cells in tissue engineering. Conducting polymers with excellent biocompatibility act as ideal tissue interface material because they can be facilely fabricated into multiple structures, patterned to undergo electrical stimulation and modified with different dopants. Meanwhile, the performance of conducting polymers is significantly influenced by the characteristics of negatively charged dopants. Herein, six kinds of biotic and abiotic molecules with electronegative groups are used as counterions to dope poly(3,4-ethylenedioxythiophene) (PEDOT). A comprehensive evaluation of the properties of PEDOT, including electrochemistry, electrical stimulation, stability and biocompatibility is provided for further comparison and analysis. This work would reinforce our understanding of the dopant-dependent performance of conducting polymers for tissue engineering and applications in electrophysiological recording and stimulation.