Background: The demand for point-of-care testing (POCT) devices has rapidly grown since they offer immediate test results with ease of use, makingthem suitable for home self-testing patients and caretakers. However, the POCT development has faced the challenges of increased cost and limited resources. Therefore, the paper substrate as a low-cost material has been employed to develop a cost-effective POCT device, known as "Microfluidic paper-based analytical devices (mu PADs) ". This device is gaining attention as a promising tool for medicinal diagnostic applications owing to its unique features of simple fabrication, low cost, enabling manipulation flow (capillary-driven flow), the ability to store reagents, and accommodating multistep assay requirements. Objective: This review comprehensively examines the fabrication methods and device designs (2D/3D configuration) and their advantages and disadvantages, focusing on updated mu PADs applications for motif identification. Methods: The evolution of paper-based devices, starting from the traditional devices of dipstick and lateral flow assay (LFA) with mu PADs, has been described. Patterned structure fabrication of each technique has been compared among the equipment used, benefits, and drawbacks. Microfluidic device designs, including 2D and 3D configurations, have been introduced as well as their modifications. Various designs of mu PADs have been integrated with many powerful detection methods such as colorimetry, electrochemistry, fluorescence, chemiluminescence, electrochemiluminescence, and SER-based sensors for medicinal diagnosis applications. Conclusion: The mu PADs potential to deal with commercialization in terms of the state-of-the-art of mu PADs in medicinal diagnosis has been discussed. A great prototype, which is currently in a real-life application breakthrough, has been updated.