Thylakoids contain photosynthetic proteins and behave as soft nanoparticles. We extracted the thylakoid from spinach chloroplasts and organized them in a film with either insulating polyethyleneimine (PEI) or conducting polyaniline (PANI) by layer-by-layer (LBL) assembly. We could achieve the improved photoelectrochemical activities from the nanostructured composite films with stable charge balanced LBL multilayers compared to the randomly deposited films. The thylakoid LBL films continuously generated photochemical electrons for longer than 130h with the 2,6-dichlorophenolindophenol (DCPIP) mediated photosynthetic energy conversion cycle. Also, the cytocompatibility of the developed thylakoid LBL films was demonstrated by PC12 neural cell attachment and differentiation tests.