In this paper, the effect of the substrate on wireless sensor network (WSN) node’s RF performance is studied experimentally by using different substrate materials with different thickness. A six-layer FR4 substrate PCB WSN node is fabricated and compared with the original two-layer FR4 PCB node to show the impact of substrate material thickness. Also different substrate dielectric constants’ impacts are studied by the same method. All these demonstrators are modeling by RF circuit analysis method and simulated in the Ansoft Designer software. Simulation results match the experimental measurement. An optimization method based on simulation for WSN node design with different substrate is presented. This analysis, modeling, simulation and optimization procedure can be carried out on some novel substrate materials such as LTCC and LCP.