Photothermal therapy with minimal invasiveness and high selectivity has been regarded as a powerful technique for tumor therapy to overcome the risks of toxic side effects and limited therapeutic efficacy of clinic cancer treatments. Among various photothermal therapeutic agents, polypyrrole (PPy) nanoparticles show a promising prospect in tumor ablation in vivo due to their admirable biocompatibility and outstanding photothermal performance. Besides, polypyrrole nanoparticles are extensively applied in biosensors, electrochemical sensors, tissue engineering, flexible microelectronics, and so on. However, the available synthesis methods of PPy nanoparticles are all time-consuming and seriously hindered their highly efficient production for diverse applications. Here we present a microwave-assisted strategy for the fabrication of PPy nanoparticles in 2 min, and the required synthesis time is shortened by 120-720 times compared to that in traditional ways. The prepared PPy nanoparticles possess uniform size, favorable aqueous solubility, and enhanced photothermal performance derived from the stronger near-infrared absorbance. Low cytotoxicity and in vivo toxicity of the nanoparticles were confirmed via comprehensive assessments. The PPy nanoparticle-based photothermal therapy in vitro led to a remarkable death of tumor cells, and in vivo tumor ablation using the nanoparticles was achieved under mild laser irradiation with a FDA-approved safe power. The proposed microwave-assisted synthesis strategy opens up a facile and ultrafast way for the construction of organic nanoparticles and facilitates a wide range of applications of them in biomedicine and other fields.