Increasing demands for the sensitive, selective, visual, conveniently solid-phase-sensing film for disease-related biomarkers, electrospun nanofibrous films (ENFFs) are a novel sensing paltform because of the high surface area ratios, networked structures, and facile functionalization. In this research, carbon dots (CDs) and quantum dots (QDs) were doped inside and electrostatically assembled on electrospun nanofibers (ENFs), affording ratiometrically dual-emitting fluoresecent films. Because of the FRET process between CDs and QDs on NFs and the strong quenching response of QDs to Cu2+, the as-prepared NFs can visually detect Cu2+ with high sensitivity and selectivity based on ratiometric fluorescent signals. By exploiting the release of numerous Cu2+ from CuO nanoparticles (NPs) under acidic conditions, a sandwich-type immunoassay with CuO NPs-labeled antibody was developed for the detection of biomarker proteins via the response of dual-emitting and FRET-based NFs to different Cu2+ concentrations. Taking advantage of the ratiometrically fluorescent property of these ENFFs as well as the Cu2+-mediated signal amplification strategy, alpha fetoprotein can be detected with high sensitivity (detection limit of 8.3 pg/mL) and selectivity. This strategy demonstrates a promising candidate with a point-of-care assay for clinical diagnostics and biomedical research.