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Designing and screening highly efficient and cost-effective luminophores have always been a challenge to develop sensitive electrochemiluminescence (ECL) biosensors. Herein, polyethyleneimine nanoparticles (PEI NPs), a kind of nonconjugated polymer (NCP) NPs with tertiary amine clusters, were developed as an ECL luminophore. Specifically, PEI NPs were synthesized by a one-step hydrothermal method using PEI and formaldehyde. The properties of PEI NPs were investigated in detail using photochemical and electrochemical techniques. The results showed cluster-dominated luminescence of tertiary amines in PEI NPs via “through-space conjugation”. This non-negligible ECL performance (at 631 nm) was also verified by the initiated reduction–oxidation process. With persulfate as a coreactant, PEI NPs acted as both the luminophore and coreaction accelerator to enhance the ECL intensity remarkably, which was eightfold higher than that of isolated PEI. Moreover, choosing dopamine as the model target, a highly sensitive “signal off” ternary ECL sensor was constructed utilizing PEI NPs as the luminophore. Dopamine could be oxidized to benzoquinone at the sensing interface, quenching the signal via ECL energy transfer. Free from any signal amplification, the proposed sensor achieved a low detection limit (4.3 nM) for target monitoring with good selectivity and stability. This strategy not only provides a unique perspective for designing novel efficient and facile ECL luminophores of tertiary amines but also broadens the biological application of NCP NPs.

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