Photothermal therapy is a new non-invasive treatment method, which can make up for the deficiency of traditional tumor therapy. As an efficient photothermal agent, carbon nanomaterials show great potential in the photothermal therapy of tumor. In this study, a rapid polymerization of pyrogallic acid and formaldehyde was induced by ultrasound-assisted method. Carbon spheres with good biocompatibility and high photothermal conversion efficiency were quickly prepared by subsequent calcination treatment. The prepared carbon spheres are highly monodisperse and uniform in size. Under 808 nm near-infrared light irradiation, they exhibit good photothermal effect and photothermal stability with photothermal conversion efficiency up to 41.4%. Cell experiments show that carbon spheres have no obvious cytotoxicity, and can effectively kill the tumor cells under near-infrared light irradiation. This work is expected to construct efficient photothermal carbon spheres for photothermal therapy of tumor.