Aging effect on the mobility and bioavailability of copper (Cu) was investigated using a spiked soil with different incubation periods from 3 to 56 d. Wheat was planted and earthworm was cultured separately in the incubated soils. The mobility of Cu in soil was evaluated by a chemical fractionation scheme and the toxicity and bioavailability were assessed by measuring the biomass and Cu concentration in tissues. Results showed that aging had a significant effect on Cu fraction distribution, of which Cu tended to incorporate from the exchangeable into more stable fractions such as the reducible and oxidisable fractions. However, aging had little effect on Cu bioavailability to wheat and earthworms. Comparing the soil being incubated for 3 d and 56 d, Cu concentration in wheat roots decreased from 14.5 to 12.8 mg/kg, and no significant changes in Cu concentration were observed in both wheat shoots and earthworms. The Cu concentration was around 2.0 and 50 mg/kg for wheat shoots and earthworms, respectively, irrespective of soil incubation time. The CaCl2-extractable Cu had a linear relationship with Cu concentration in wheat roots (R2 = 0.65, P < 0.05), but no linear relationship can be found for wheat shoots and earthworms. Biological control may be more crucial for Cu accumulation in organism than the changes in soil Cu fraction caused by aging.