Drop-on-demand microkits with a diameter of ∼20 μm are used to measure the activity of acetylcholinesterase (AChE) in a brain slice with single-cell resolution. The relative standard deviation from 25 cellular regions reached 73.3% exhibiting the difference of enzyme activity in the brain slice. Therefore, this approach utilizing the well-established kits provides an alternative single-cell-resolved strategy for the elucidation of enzymatic heterogeneity at the tissue level.