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A novel microfluidic system which is fabricated with five polydimethylsiloxane layers for interstitial fluid (ISF) extraction, collection, and measurement toward the application of continuous and real-time glucose monitoring is presented in this paper. The system consists of a micro vacuum generator for ISF transdermal extraction and fluid manipulation, micro chambers for the collection of ISF, micro pneumatic valves for fluid management, and a micro flow sensor for ISF volume measurement. Sequentially controlled by the pneumatic valves, the ISF extraction, collection, and volumetric measurement functions of the system were demonstrated using the stable vacuum generated by the integrated vacuum generator. Through low-frequency ultrasound pretreated full-thickness pig skin, the normal saline solution with different glucose concentrations was transdermally extracted, collected, and measured. The absolute error in the volume measurement of the transdermally extracted “ISF analog” was less than 0.05 μL. The microfluidic system makes it possible to realize the clinical application of continuous glucose monitoring based on ISF transdermal extraction technology.