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Worldwide, two million two hundred thousand new cases of breast cancer were diagnosed in 2020. Traditional breast cancer treatments have several side effects, such as poisoning of the heart and lung during chemotherapy and cardiovascular disease during radiotherapy. Moreover, many patients suffer from acute and chronic pain, loss of breast shape, and limited arm mobility after surgery. Researchers strive to reduce adverse effects by finding more effective strategies. Sonodynamic therapy is a newly developed cancer treatment that offers safety, high penetration depth into soft tissues, accessibility, affordability, etc. In addition to these benefits, sonodynamic therapy also enhances chemotherapy effectiveness. Therefore, the combination of sonodynamic therapy and chemotherapy along with a multifunctional sensitizer can enhance treatment effectiveness while minimizing side effects. In this study, PEGylated gold nanoparticles (PEG-GNPs) were synthesized and loaded with an anticancer agent, Gemcitabine (Gem-PEG-GNPs). The synthesized Gem-PEG-GNPs act as sonosensitizers and nanocarriers, which improve the efficacy of sonodynamic therapy and chemotherapy as well as reduce harmful effects. Ultrasound waves combined with Gem-PEG-GNPs increased apoptosis and decreased survival rates in MCF-7 cells. When Gem-PEG-GNPs and ultrasound waves were combined, a significant synergistic effect was observed compared to ultrasound waves alone. In conclusion, Gem-PEG-GNPs have the potential to be effective sonosensitizers and drug delivery agents in treatment of breast cancer.