The use of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs) was studied. Specific chemical features of the nanofibers were achieved by doping the base polymer, poly(lactic acid) (PLA), with poly(ethylene glycol) (PEG) and polystyrene8K-block-poly(ethylene-ran-butylene)25K-block-polyisoprene10K-Brij76 (K3-Brij76) (KB). The LFAs were assembled such that the sample flowed through the nanofiber mat via capillary action. Initial investigations focused on the sustainable spinning and assembly of different polymer structures to allow the LFA format. Here, it was found that the base polymer poly(vinyl alcohol) (PVA), which was shown to function well in microfluidic biosensors, did not work in the LFA format. In contrast, PLA-based nanofibers enabled easy assembly. Three relevant features were chosen to study nanofiber-based functionalities in the LFA format: adsorption of antibodies, quantification of results, and nonspecific binding. In particular, streptavidin-conjugated sulforhodamine B (SRB)-encapsulating liposomes were captured by anti-streptavidin antibodies adsorbed on the nanofibers. Varying the functional polymer concentration within the PLA base enabled the creation of distinct capture zones. Also, a sandwich assay for the detection of Escherichia coli O157:H7 was developed using anti-E. coli antibodies as capture and reporter species with horseradish peroxidase for signal generation. A dose-response curve for E. coli with a detection limit of 1.9 × 10(4) cells was achieved. Finally, functional polymers were used to demonstrate that nonspecific binding could be eliminated using antifouling block copolymers. The enhancement of paper-based devices using functionalized nanofibers provides the opportunity to develop a broad spectrum of sensitive and specific bioassays with significant advantages over their traditional counterparts.