A long-term field experiment was set up on a Luvisol with various fertiliser treatments including bio-compost. The effect of compost application on total (aqua regia extractable) Cu and Zn contents in soil layers were investigated. After 12 years compost application, organic fractions considered as bioavailable and less accessible inorganic Cu and Zn fractions had altered compared to the treatments without compost application. In comparison to the topsoil, the subsoil aqua regia extractable Cu and Zn were higher in the mineral fertilisation treatment (topsoil: 18.46 mg Cu kg−1, 62.89 mg Zn kg−1; sub-soil: 19.89 mg Cu kg−1, 66.88 mg Zn kg−1), but lower in the compost treatment (topsoil: 22.58 mg Cu kg−1, 70.80 mg Zn kg−1; subsoil: 21.03 mg Cu kg−1, 68.68 mg Zn kg−1). In compost, Cu was mainly found in less accessible inorganic fractions and a smaller proportion in the organic fraction. In contrast, the organic fraction was most dominant for Zn followed by the less accessible inorganic fractions. Only a non-drastic accumulation of Cu and Zn in soil was detected. Cu and Zn fractionations in composts had the same trend of the corresponding fractions in the soil.