The use of low-cost, available, biodegradable, and biocompatible materials is very important in the design and preparation of biosensors. Nanopapers are an appropriate candidate as the substrate in the design and preparation of high-performance biosensors due to their good properties such as high dielectric constant, insulating properties suitable for thin-film formation, lamination properties, biocompatibility, and biodegradability. Nanopaper-based biosensors can be discussed in two perspectives: diagnosis and device development. They have been developed in two categories of nanopaper-based lateral flow test strips and microfluidic nanopaper-based analytical devices. On the other hand, several techniques were used as the quantitative diagnostic methods in nanopaper-based biosensors such as colorimetry, electrochemiluminescence, photoluminescence, fluorescence, surface-enhanced Raman scattering (SERS), and electrochemical techniques. This chapter has tried to briefly discuss the diagnostic methods in nanopaper-based biosensors and their performance.