Three dimensional (3D) cell culture in functional scaffolds to mimic the cell natural growth state is important for the construction of cell based implants in vitro for tissue engineering applications. Herein, we report a novel fluffy polypyrrole (PPy) fibrous scaffold (fluffy-PPy scaffold) fabricated by means of an improved electrospinning process combined with in situ surface polymerization, in which PPy hollow fibers are discrete from one another with deep interconnected pores of similar to 100 mu m. This unique spatial structure permits the easy entry of cells into the fluffy-PPy scaffold with no extra help to achieve complicated 3D cell culture methodologies. The cell proliferation and morphology of cardiomyocytes (as a model cell) cultured in the fluffy-PPy scaffold were tested over a 3 day culture period. Evidence was provided that cardiomyocytes entered into the interior of the fluffy-PPy scaffold and formed stable cell-fiber constructs, and the rate of cell proliferation was higher than that on a traditional electrospun PPy fibrous mesh (mesh-PPy scaffold) and tissue culture plates (TCP). These results demonstrate that the fluffy-PPy scaffold not only achieved 3D cell culture, but also resulted in increased cell proliferation. Therefore, we suggest that the fluffy-PPy scaffold may be an appropriate choice as a functional scaffold capable of supporting 3D cell culture in the field of cardiac tissue engineering.