Novel multilayer hollow microspheres were fabricated via the layer-by-layer (LBL) assembly technique by the electrostatic interaction between the polycation chitosan (CHI) and the anion carboxyl-fiinctionalized multiwalled carbon nanotubes (f-MWNTs) on sacrificial polystyrene sulfonate (PSS) microsphere templates. The successfal stepwise growth of the CHI/f-MWNTs multilayers in different pH media was compared by zeta potential measurement and scanning electron microscopy (SEM) analysis. The morphologies of the hollow microspheres were characterized by transmission electron microscopy (TEM) and SEM. The swelling and collapse of the hollow microspheres occurred during the template removal by dissolution and the drying process. It was found that more f-MWNTs had been deposited onto the surface of the PSS microspheres at pH 3 than at pH 5, confirmed by SEM and thermogravimetric analysis (TGA). Furthermore, this result is consistent with the differences of the electrical conductivities of the hollow microspheres obtained from the different pH media. It is also found that in the etching process, the different etching reagents, such as DMF and toluene, obviously affected the electrical conductivity of the final hollow microspheres.