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Ore particle segregation commonly occurs during dump leaching because of the differences of particle size, surface roughness, and relative density. The presence of a fine interlayer is key factor of the uneven erosion of the ore surface during leaching, which seriously limits the bioleaching efficiency of copper extraction. To explore the interaction effects of fine interlayers on leaching behavior, the surface morphology and passivation occurrence during the leaching process was studied, where coarse ore particles (4 mm < d < 6 mm) and fine ore particles (2 mm < d < 4 mm) were selected, and a bioleaching experiment of secondary copper sulfide with fine interlayers located at different positions was carried out. Analysis were carried out using computed tomography (CT) scanning technology and field-emission scanning electron microscopy-energy dispersive spectrometry (FE SEM-EDS) technology in the macro, meso, and micro scales. As a result, the macro leaching dynamics, meso-scale ore particle agglomeration, and micro surface morphology characteristics as well as passivation were studied. The results show that fine interlayer leads to a lower copper extraction rate, which is lower than when the fine interlayers are mixed with homogeneous coarse granular medium. The effects of fine interlayers on ore extraction depend on their location. In the experiment, the fine interlayers located at the top results in the highest copper extraction rate (71.3%) after leaching for 60 days; the degrees of evolution of the ore surface pore structure are different at different heights inside the same fine interlayers. The copper extraction rate reaches its peak after leaching for 60 days. The ore particle agglomerations and passivation phenomenon are significant. Passivation layers, such as of jarosite, polysulfide, extracellular polymeric substances, sulfur film, are formed on the ore surface.