In this study, a more efficient, convenient, and cost-effective way was proposed to design and fabricate a polydimethylsiloxane (PDMS) microfluidic device using 3D printing and transfer techniques. Typically, sensor measurements can only be conducted by immersing the sensor in a solution under static conditions. However, in this study, a newly designed microfluidic device combined with a microfluidic system enables dynamic measurements with sensors. By controlling the flow and temperature of the test liquid, the physiological environment of the human body under various conditions can be simulated, resulting in more accurate and realistic data. During the dynamic condition, an average sensitivity of 6.396 mV/(mg/dL) and linearity of 0.999 were obtained at a flow rate of 75.0 μL/min. When the temperature was at 37 • C, the biosensor achieved an average sensitivity of 4.381 mV/(mg/dL) and a linearity of 0.999. The dynamic response time was 7 seconds, and the drift rate was 1.47 mV/hr. The experimental results obtained under dynamic conditions showed a significant improvement in the sensing characteristics of the urea biosensor compared with the results obtained under static conditions.