Food standards control copper in water and several foods by setting ADI values. The metal, an essential element influencing biochemical functions, is involved in oxidative stress resistance. However, high metal concentrations can be toxic, especially in nerve functions. Copper at 1 and 2 mM could toxic generations of Drosophila melanogaster flies by assessing the development, learning, memory, locomotion, activities of catalase and acetylcholinesterase, and the neutralizing DPPH-free radicals. Flies cultured in a copper-containing medium lengthened their maturation period and reduced hatching numbers. The learning ability in larvae and the climbing ability of adult flies also decreased between generations. The antioxidative capacity showed the differences between fly groups. Catalase activity gradually decreased with each generation, while acetylcholinesterase did not indicate profound differences between generations. In conclusion, a daily intake of 1–2 mM copper could affect fruit flies’ behaviors, nervous system functions, and the benchmark dose of copper in food.