Microfluidic channels fabricated over fabrics or papers have the potential to find substantial application in the next generation of wearable healthcare monitoring systems. The present work focuses on the fabrication procedures that can be used to obtain practically realizable fabric-based microfluidic channels (μFADs) utilizing patterning masks and wax, unlike conventional printing techniques. In this study, comparative analysis was used to differentiate channels obtained using different masking tools for channel patterning as well as different wax materials as hydrophobic barriers. Drawbacks of the conventional tape and candle wax technique were noted and a novel approach was used to create microfluidic channels through a facile and simple masking technique using PVC clear sheets as channel stencils and beeswax as the channel barriers. The resulting fabric based microfluidic channels with varying widths as well as complex microchannel, microwell, and micromixer designs were investigated and a minimum channel width resolution of 500 μm was successfully obtained over cotton based fabrics. Thereafter, the PVC clear sheet-beeswax based microwells were successfully tested to confine various organic and inorganic samples indicating vivid applicability of the technique. Finally, the microwells were used to make a simple and facile colorimetric assay for glucose detection and demonstrated effective detection of glucose levels from 10 mM to 50 mM with significant color variation using potassium iodide as the coloring agent. The above findings clearly suggest the potential of this alternative technique for making low-cost and practically realizable fabric based diagnostic devices (μFADs) in contrast to the other approaches that are currently in use.