A simple method for making two-dimensional (2D) protein arrays using a new spreading technique was developed. A solution of the iron storage protein ferritin was injected into an aqueous subphase that had a higher density and surface tension than protein solution. The buoyancy of the protein solution made it rise to the surface where it spread quickly and smoothly. For the stage of rising and spreading, the buoyancy and surface tension were critical factors. The proteins that reached the air-water interface unfolded instantaneously and formed a continuous film. These films were transferred onto holey carbon films for transmission electron microscopic analysis. The holes of the carbon film were found to be covered with a smooth, amorphous film of unfolded protein onto which intact ferritin was adsorbed. For the stage of 2D array formation, the presence of cadmium ion in the subphase was a critical factor. The 2D array formation occurred in the thin layer of protein solution caught between the unfolded protein film of the surface and the subphase below.