Point-of-care (POC) antigen detection plays a crucial role in curbing the spread of viruses. Paper-based fluorescence aptasensors are expected to offer a low-cost tool to meet the needs of decentralized POC diagnosis. Herein, we report on a fluorescent paper-based sensing system for detecting the SARS-CoV-2 spike protein. The sensing system was constructed by loading multi-layer Nb₂C MXene nano-quenchers and carbon-dot-labeled aptamer (G-CDs@Apt) probes onto a mixed cellulose ester (MCE) paper substrate. On the Nb₂C MXene/G-CDs@Apt sensing paper, abundant G-CDs@Apt probes were attached to the multilayer MXene nano-quenchers and kept in a fluorescence-off state, while recognition of the target detached the G-CDs@Apt probes formed the nano--quenchers, resulting in fluorescence recovery of the sensing paper. The developed paper-based sensor performed well in the one-step detection of the SARS-CoV-2 S1 protein with a detection limit of 0.067 ng/mL (0.335 pg/test). The assay exhibited good selectivity and anti-interference in the detection of the SARS-CoV-2 S1 protein in artificial saliva. Moreover, the paper-based aptasensor was successfully used to detect the SARS-CoV-2 S1 protein in actual environmental samples with recoveries of 90.87–100.55% and relative standard deviations of 1.52–3.41%. The proposed technology provides a cost-effective alternative to traditional antibody test strips for a wide range of POC diagnostic applications.