Terminal　deoxynucleotidyl　transferase　(TdT)　is　upregulated　in　several　types　of　leukemia　and　is　considered　a　disease　biomarker　and　a　potential　therapeutic　target　for　leukemia.　In　this　research,　a　homogeneous　electrochemiluminescence　(ECL)　method　based　on　the　control　of　surface　charge　and　morphology　of　tris　(2,2′-bipyridine)　ruthenium(II)　chloride　hexahydrate-doped　silica　nanoparticles　(Ru@SiO2　NPs)　has　been　designed　for　TdT　activity　detection.　A　small　amount　of　short　single-stranded　DNA　(ssDNA)　was　modified　onto　the　surface　of　Ru@SiO2　NPs,　and　the　nanoparticles　with　a　slight　positive　charge　experienced　electrostatic　attraction　with　the　indium　tin　oxide　(ITO)　electrode　with　a　negative　charge,　so　relatively　high　ECL　signals　had　been　detected.　Under　the　action　of　TdT,　the　ssDNA　was　significantly　elongated,　carrying　numerous　negative　charges　on　its　phosphate　backbone,　so　the　overall　negative　charge　of　the　reporter　nanoparticles　was　enhanced,　resulting　in　a　strong　electrostatic　repulsion　with　the　ITO　electrode.　Simultaneously,　the　long　ssDNA　wrapped　around　the　nanoparticles　hindered　the　approach　of　the　coreactant.　Due　to　the　dual　effects,　the　ECL　response　of　the　system　decreased.　The　constructed　biosensor　exhibited　excellent　sensitivity　toward　TdT　over　a　range　spanning　from　1　to　100　U/L.　The　limit　of　detection　is　as　low　as　1.78　U/L.　The　developed　approach　was　effectively　applied　to　detect　TdT　activity　in　leukemic　patients’　leukocyte　extracts.　©　2023　American　Chemical　Society.