Electrofluidics is the unique combination of electrophoresis and microfluidics, which has opened up broad opportunities for bioanalysis and multiplexed assay. These systems typically comprise inaccessible and fully enclosed microcapillary or microchannels, with limited sample loading capacities and no direct access to the solutes within. Here, we investigate the application of multiyarn textile assemblies which provides an open and surface accessible electrophoretic separation platform. Three-dimensional (3D) textile structures have been produced using conventional knitting and braiding techniques from a range of commercially available yarns. Capillary zone electrophoresis separation studies have been carried out on these substrates using fluorescent anionic (fluorescence, FL) and cationic (rhodamine-B, Rh-B) markers. The effects of different yarn surface chemistry, textile fabrication technique, and electrolyte ionic strength on the electrophoretic mobility of the test analytes have been studied. From the broad range of yarns investigated, polyester was shown to have the highest electrophoretic mobility for Rh-B (6 × 10-4 cm2 V-1 s-1) and for FL (4 × 10-4 cm2 V-1 s-1). The braiding approach, being simple and versatile, was found to be the most effective route to produce 3D textile-based structures and offered the potential for selective movement and targeted delivery to different channels. Composite braids made with yarns of differential surface chemistries further revealed a unique behavior of separation and parallel movement of oppositely charged ionic species. We also demonstrate the feasibility to apply isotachophoresis (ITP) on these braided textile substrates to rapidly focus dispersed FL sample bands. Here, we demonstrate the focusing of FL from a dispersed band into narrow band with a 400 times reduction in sample width over 90 s. Owing to the simplicity and reproducibility of the developed approach, textile-based inverted microfluidic applications are expected to enable opportunities in bioanalysis, proteomics, and rapid clinical diagnostics.