Textile-based sensors fabricated using the direct-coating method are the appropriate choice to meet the aspects of flexibility, non-invasiveness, and lightness for continuous monitoring of the human body. The characteristics of the sensor substrate are directly influenced by factors such as the type of weave, thread fineness, fabric density, and the type of polymeric constituent fibers. The fabric used as the sensor substrate, fabricated using the direct-coating method, must be capable of retaining the electrode paste solution, which has higher viscosity, on one surface of the fabric to avoid short circuits during the fabrication process. However, during its application, this fabric should allow the easy passage of analyte solutions with low viscosity as much as possible. Hence, an appropriate fabric construction is required to serve as the substrate for textile-based sensors to ensure the success of the fabrication process and the effectiveness of the resulting sensor’s performance. The development of the structural design of the fabric to be used as a substrate for non-invasive biosensors with a multilayer concept is carried out by weaving and sewing processes utilizing polyester-viscose fibers. During the production process, variations are applied, such as weft yarn density, the characterization of wetting time, absorption rate, maximum wetted radius, spreading speed, and accumulative one-way transport index. The most suitable fabric for use as a substrate for non-invasive biosensors with a multilayer concept, such as in this research, is a fabric with a weft thread density of 70 strands per inch, along with the addition of an analyte transfer thread configuration.