The discovery and function analysis of terminal deoxynucleotidyl transferase (TdT) add a new dimension to the understanding of leukemia mechanisms and stimulate the development of new analytical tools for leukemia diagnosis. Herein, taking advantage of the inherent property of TdT for performing DNA synthesis using only single-stranded DNA as the nucleic acid substrate, we developed a self-customized multichannel exponential amplification (SMEA) system for the fluorescent sensing of TdT activity. The SMEA design employs an intermolecular DNA interaction made of a nicking site-incorporated elongation primer (EP) and a nicking site-incorporated poly-thymine tailed molecular beacon (Poly-T-MB). The absence of TdT is unable to bridge the relationship between EP and Poly-T-MB, ensuring the SMEA has an ultralow background. The presence of TdT, however, leads to the elongation of EP to poly-adenine tailed EP (Poly-A-EP) under a dATP pool responsible for further hybridization with numerous Poly-T-MB. With the aid of polymerase and nickase to react with the hybridization product of Poly-A-EP/(Poly-T-MB)n, it can cause bidirectional strand nicking, polymerization, and displacement in many cycles and channels. In this case, the SMEA is found to be associated with the configuration transformation and splitting of all Poly-T-MBs for a significant fluorescence enhancement. Depending on this high target signal amplification and strong background inhibition abilities, the SMEA sensing system is powerful for the qualitative and quantitative determination of TdT activity, showing that it has great promise for biomedical study and disease diagnosis.