In this study, we present the multilayered symmetrical droplet splitting microfluidic system for preparation of microspheres. The microfluidic device was fabricated by conventional photolithography and PDMS casting. Multiple layers of microfluidic channels for symmetrical droplet splitting were stacked and integrated into a device. Each layer was designed to obtain 16 microdroplets from one droplet by droplet splitting. The droplet size was controlled with flow rate of dispersed phase (DI-water) and continuous phase (Mineral Oil with 3 wt.% SPAN80) by using a syringe pump. The droplet splitting behavior and production rate were analyzed by high-speed camera and inverted microscope in one layer of the microfluidic device. Additionally, the droplet size and size distribution were observed in each layer of the microfluidic device. The droplet size could be controlled by flow control of two phase flows with high uniformity of droplet size less than 5% coefficient of variation.