Paper-based analytical devices (PADs) and Electrospun Fiber-Based Biosensors (EFBs) have aroused the interest of the academy and industry due to their affordability, sensitivity, ease of use, robustness, being equipment-free, and deliverability to end-users. These features make them suitable to face the need for point-of-care (POC) diagnostics, monitoring, environmental, and quality food control applications. Our work introduces new and experienced researchers in the field to a practical guide for fibrous-based biosensors fabrication with insight into the chemical and physical interaction of fibrous materials with a wide variety of materials for functionalization and biofunctionalization purposes. This research also allows readers to compare classical and novel materials, fabrication techniques, immobilization methods, signal transduction, and readout. Moreover, the examined classical and alternative mathematical models provide a powerful tool for bioanalytical device designing for the multiple steps required in biosensing platforms. Finally, we aimed this research to comprise the current state of PADs and EFBs research and their future direction to offer the reader a full insight on this topic.