Objective To avoid the accumulation of copper sulfide (CuS) nanoparticles, prepare and optimize CuS nanoparticles, analyze the factors affecting the particle size and evaluate their photothermal properties. Methods Based on the single factor study, central composite design-response surface methodology was used to optimize the CuS nanoparticle formulation process. The morphology, particle size stability, photothermal conversion efficiency, photothermal stability of optimized CuS nanoparticles were characterized. The toxicity of CuS nanoparticles on 4T1 breast cancer cells and HK2 kidney cells was evaluated by CCK-8 method. In vitro photothermal experiment was used to investigate the ability of CuS nanoparticles on killing 4T1 breast cancer cells. Results The average hydration dynamic diameter of optimized CuS nanoparticles was (10.53±1.63)nm, the actual particle size of CuS nanoparticles showed by TEM image was (3.10±0.81)nm. It had good particle size stability, good photothermal conversion efficiency and photothermal stability. Within the concentration range of 100 μg/ml and 150 μg/ml，it showed no significant toxicity on 4T1 breast cancer cells and HK2 kidney cells, indicating the good stability of CuS nanoparticles. In vitro photothermal therapy showed that CuS nanoparticles had good ability to kill 4T1 breast cancer cells by photothermal. Conclusion The prepared CuS nanoparticles have a small particle size (less than 6nm) and a good photothermal effect, which is expected to solve the problem of CuS nanoparticles accumulation in vivo and make it better for tumor treatment.