Nanotwinned copper has excellent properties of high strength, high conductivity and high toughness, which makes it a research hotspot in the field of electronic packaging in recent years. During the electrodeposit procedure, the additives play a vital role in the quality and property of nanotwinned copper. Among these additive, methylene blue is commonly used in the direct-current (DC) electroplating regular copper industry. In this study, the effect of methylene blue on the microstructure and mechanical property of nanotwinned copper was investigated. It was found that the microscopic morphology and mechanical properties of nanotwinned copper are closely related to the concentration of methylene blue. As the concentration increases, the growth rate of electrodeposits shows a decreasing tendency, demonstrating a strong grain refinement effect. When the methylene blue concentration is 2 mg/L, columnar grains start growing from the bottom substrate to upper surface, and there exists a high-density twin structure within the columnar grains. What’s more, the tensile strength of the film at this concentration is 194 MPa, which is almost twice as that of regular coarse-grain copper (about 110 MPa) as measured by the dynamic thermomechanical analyzer (DMA). The surface hardness of the film can reach 1.6 GPa, which is also higher than the hardness of coarse-grained copper.