Paper has been a popular material of choice for biomedical applications including for bioanalysis and cell biology studies. Regular cellulose paper-based devices, however, have several key limitations including slow fluid flow; large sample retention in the paper matrix for microfluidic paper-based analytical device (μPAD) application; serious solvent evaporation issues, and contamination and poor control of experimental conditions for cell culture. Here, we describe the development of two novel platforms, nanopaper-based analytical devices (nanoPADs) and nanofibrillated adherent cell-culture platforms (nanoFACEs), that use nanofibrillated cellulose (NFC) paper, simply called nanopaper, as the substrate material to create transparent, pump-free and hollow-channel paper-based microfluidic devices. Due to the natural hydrophilicity and nanoscale pore size of nanopaper, the hollow-channel microfluidic devices can realize a totally pump-free flow without any complicated surface chemical functionalization on the nanopaper. Experimental results showed that within a certain range, larger hollow channel size leads to faster pump-free flows. Different from previous designs of paper-based hollow-channel microfluidic devices, the high transparency of the nanopaper substrate enabled the integration of various optical sensing and imaging technologies together with the nanoPADs and nanoFACEs. As proof-of-concept demonstrations, we demonstrated the use of nanoPADs for colorimetric sensing of glucose and surface-enhanced Raman spectroscopy (SERS)-based detection of environmental pollutants and applied the nanoFACEs to the culture of human umbilical vein endothelial cells (HUVECs). These demonstrations show the great promise of nanoPADs and nanoFACEs for biomedical applications such as chemical/bioanalysis and cell biology studies.