This paper presents a system that picks up protoplasts produced by removing the surrounding cell wall of root cells while preserving their positional information. The fundamental concept of this system involves scanning the root tip over time using a confocal microscopy to measure the positional information of each cell. Then, the protoplast pickup is conducted after switching to a brightfield microscopy to ensure the certainty of pickup. The system measures the position of single protoplasts, adjusts the position of the pipette using a 3-axis micromanipulator, and picks up the target protoplast using a microfluidic pump driven by a piezoelectric actuator. To automate this pickup process, we achieved calibration of the system. The fully automatic 3D calibration of the pipette tip was achieved, allowing 3D micromanipulation under the microscope with an accuracy of 3.1 μm in the XY-plane. Furthermore, by implementing multiple functions such as automatic detection of protoplasts, the process of protoplast pickup has been achieved.