By using poly-L-lysine (PLL) as reducing agent and stabilizer, a novel method for synthesizing gold nanoparticles (GNP) under mild temperature conditions was reported. The morphology and size of these GNP could be easily controlled by changing the amount of PLL and temperature. And the GNPs prepared by this method had better stability than traditional methods. On this basis, the GNPs' biological activity was studied in detail using breast cancer cells MCF-7 as object. Experimental results indicated that the GNPs prepared by PLL had better endocytosis efficiency and higher proliferation inhibition rate than which prepared by traditional method. This method has valuable property for the application of GNPs in biomedical engineering.