Conventional transverse relaxation time (T2)-mediated magnetic resonance sensors (MRS) that utilizing the target-induces state change of magnetic nanoparticles (MNPs) mainly suffer from low sensitivity. Recent T2-MRS that based on target-induced amount change of MNPs can achieve a higher sensitivity, but these sensors can hardly accommodate small molecules. We herein develop an ultrasensitive T2-MRS that enable the detection of small molecules based on cascade bioorthogonal reactions (BRs)-realized MNPs binding and assembly. Benefiting from rapid and highly selective cascade BRs, a single small molecule target can not only increase MNPs binding but also assembly MNPs, which greatly amplifies T2 signal for sensing based on both the state and amount change of MNPs for the first time. Our strategy is capable of sensing chlorpyrifos with a liner range of 0.1 ng/mL to 1000 ng/mL. We justify the practicability of our assay by detecting chlorpyrifos in apple and cabbage samples, whose accuracy is higher than that of enzyme linked immunosorbent assay. Our assay provides a cascade BRs-mediated MRS that can greatly broaden the use of T2-based MRS for ultrasensitive sensing trace small molecules in complex samples.