Layer-by-layer nanostructured gamma-NaxCO2O4 hexagonal crystals have been synthesized by a novel protein adsorption method. The formation procedure of layer-by-layer nanostructured gamma-NaxCO2O4 crystals was investigated by powder X-ray diffraction (XRD), field emitted scanning electron microscopy (FESEM), and high-resolution electron microscopy (HREM). The growth mechanism of layer-by-layer nanostructured gamma-NaxCo2O4 crystals is related to the formation of colloidal precursor and the construction of the oriented layered structure. Ultrasound irradiation results in the formation of network structure colloidal precursor composed of bovine serum albumin (BSA), cobalt, and sodium sources due to the linking of BSA molecules as disulfide bonds and the electrostatic attraction forces. By a freeze-drying method, the oriented layered structure of precursor is obtained with the well-dispersed cobalt and sodium sources. At 800 degrees C, with the aid of an oriented layered structure, layer-by-layer nanostructured gamma-NaxCo2O4 hexagonal crystals are assembled by 15-30 nm nanoflaky hexagonal crystals along the perpendicular direction to the plane of nanoflaky hexagonal crystals.