Pesticide residues have seriously threatened human health and affected the sustainable development of the environment. It is pivotal to establish an efficient detection platform for pesticide assays. A novel ratiometric fluorescent aptasensor for sensitive determination of pesticide has been successfully established. Carbon dots (CDs) are used as the reference emitter, while the T-rich single-stranded DNA templated copper nanoparticles (CuNPs) provide the increasing signal. More precisely, CDs/magnetic beads (MBs) combined with split aptamer are used in this detection platform to present a label-free and enzyme-free isocarbophos (ICP) detection with high sensitivity and specificity. In the presence of ICP, a sandwich-typed complex is formed which attaches a T-rich DNA. After magnetic separation and addition of Cu/ascorbic acid (AA), the CuNPs are generated and the strong red luminescent appears. Without ICP, just blue emission from CDs exits. The developed ratiometric aptasensor offers an excellent ICP detection in the linear range of 3–200 nM with a detection limit of 0.81 nM. It provides new ways for the assay of pesticides in agricultural products or environmental samples.