The combination of optical clearing with light microscopy has a number of applications in the whole-brain imaging of mice. However, the initial processing time of optical clearing is time consuming, and the protocol is complicated. We propose a novel method based on on-line optical clearing. Agarose-embedded mouse brain was immersed in the optical clearing reagent, and clearing of the brain was achieved ~100 μm beneath the sample surface. After imaging, the cleared layer was removed, thereby allowing layer-by-layer clearing and imaging. No pre-immersion was required, and we demonstrated that on-line optical clearing can reduce the whole-brain imaging time by half.