PPY nanoparticles, as one of the important organic photothermal agents has been attracted great attention due to their good biocompatibility, high photothermal efficiency, and low cost. In order to further evaluate the size-dependent photothermal effect, PPY nanoparticles with two different sizes have been prepared with a facile method. The near infrared absorption and photothermal effect of the two size PPY nanoparticles were compared. The PPY nanoparticle with small size shows better photothermal effect at the same condition. The photostability, stability, and cytotoxicity in biologic system have also been investigated. In addition, the PPY nanoparticles with small size can kill the cancer cells effectively under the irradiation of 808 nm laser with a low-power density. These findings may provide better information for the application of the PPY nanoparticles on the photothermal ablation of cancer cells.