A 3-dimensional liquid flow control method has been developed to manipulate and retain a single yeast cell freely in a microchip. This method allows us to carry out single-cell experiments by selecting any desired single cell from a group, retaining the cell for cellular signal detection, and delivering reagents to the cell during continual detection and observation without any negative impact from the liquid flow on the live cell. The cell was scanned back and forth across an observation window in order to extract pure cellular fluorescent signals. Different scanning methods were discussed for effective collection of the cellular fluorescent signal. The cell scanning technique results in many advantages, such as distinguishing a small part of a cell, allowing for background correction and monitoring the switch of reagents. In addition, it is possible to evaluate the photobleaching effects on both the background and cellular fluorescence, with the latter found to be less significant in a restricted cellular environment.