An enzyme-free and label-free visual sensing strategy was developed for sensitively detecting thrombin using a plasmonic nanoplatform. Both the thrombin-triggered catalytic hairpin assembly (CHA) amplification reaction and G-quadruplex/hemin DNAzyme-controlled plasmonic signal readout were engineered on an electrospun nanofibrous membrane. Owing to its large specific surface area and porous structure, the nanofibrous membrane enhanced the loading capacity of B-H2 and the interface interaction efficiency. This plasmonic nanoplatform was used to perform the sensitive and naked-eye detection of thrombin as low as 1.0 pM in human serum samples. This visual strategy can discriminate thrombin from other co-existing proteins very well. Moreover, the visual sensing platform exhibited excellent reusability and long-term stability. The proposed enzyme-free and label-free plasmonic nanoplatform is low-cost, easy to operate and highly sensitive, and has potential applications in the point-of-care detection of protein biomarkers.