A carbon nanoparticle (CNP) and Cryonase-aided method that realizes the amplified fluorescent detection of theophylline was proposed. The amplification technique exploits distinct binding affinities of CNP towards the FAM-labeled theophylline RNA aptamer (aptasensor) and aptasensor/theophylline complex as well as the protection effect of CNP for absorbed aptasensor from enzymatic digestion by Cryonase. Upon the addition of theophylline, it forms an aptasensor/theophylline complex with a fluorescent dye-tagged aptasensor that is initially absorbed and quenched by CNP. The nuclease activity of Cryonase towards detached aptasensor probes is then activated, leading to efficient cleavage of aptasensor probes and separation of the fluorescent dye from the CNP surface. Theophylline that has been liberated can launch another reaction cycle, which ensures the sensitivity enhancement. A detection limit is achieved as high as 6.3 nM, which is 400-fold better than traditional strategies. The proposed sensing system also provides desired selectivity even in serum samples. The assay is simple, sensitive, selective, and universal, and has great promise for the design and application of aptasensors in the biological, chemical, and biomedical fields.