Thread-based microfluidics, which rely on capillary forces in threads for liquid flow, are a promising alternative to conventional microfluidics, as they can be easily integrated into wearable textile-based biosensors. We present here advanced textile-based microfluidic devices fabricated by machine stitching, using only commercially available textiles. We stitch a polyester “Coolmax®” yarn with enhanced wicking abilities into both hydrophobic fabric and hydrophobically treated stretchable fabric, that serve as non-wicking substrates. In doing so we construct textile microfluidics capable of performing a wide variety of functions, including mixing and separation in 2D and 3D configurations. Furthermore, we integrate a stitched microfluidic device into a wearable T-shirt and show that this device can collect, transport, and detect sweat from the wearer's skin. These can also be machine-washed, making them inherently reusable. Finally, we integrate electrochemical sensors into the textile-based microfluidic devices using stitched gold-coated yarns to detect analytes in the microfluidic yarns. Our stitched textile-based microfluidic devices hold promise for wearable diagnostic applications. This novel, bottom-up fabrication using machine stitching is scalable, reproducible, low-cost, and compatible with the existing textile manufacturing industry.