Point-of-care tests (POCT) are rapid, simple, portable, inexpensive, and convenient-to-use diagnostic tests that don’t need much introduction considering that all of us have used the lateral-flow assay (LFA) Covid-19 antigen tests over the course of the last two years. Much of the POC research field focuses on strategies to improve such LFAs with respect to their sensitivity and ability to provide quantitative results rather than only yes-or-no answers. Here, we studied a novel concept by merging LFAs with nanomaterial-enhanced electrochemical detection, which is well known for its quantitative and sensitive characteristics. Thus, we have developed a novel carbon nanofiber-based electrochemical transducer to harness its superior electrochemical performance and combine it with its fibrous characteristics. This unique property allows the flow of sample solutions directly through the transducer. The carbon nanofibers are obtained through the carbonization of electrospun polyimide nanofibers via a CO2 laser generating free-standing 3D electrodes. We demonstrate their capabilities by highly sensitive detection of silver in the nM range, integrating them into a microfluidic channel to detect the neurotransmitter dopamine in the pM region, and finally embedding them in an LFA structure for the detection of pathogenic DNA. These three examples demonstrate that the nanofiber-based electrodes offer excellent sensitivity for the detection of a broad range of analytes in a variety of assay set-ups. They are therefore of great interest for use in the POCT due to their inexpensive and simple fabrication.