A doped polymer nanoparticle (dPNP) of electrochemiluminescence (ECL) was prepared via doping the anionic polyelectrolyte polyacrylic acid (PAA) and the cationic polymer poly-ethyleneimine (PEI) into the polymer nanoparticle (PNP), which was self-assembled by Ru(bpy)32+ derivative-grafted PAA (PAA−Ru) with both cations and anions. The good electrical conductivity of the doped polyelectrolyte PAA enhanced the ECL intensity of PNP to 109.1%, and the involvement of a large number of tertiary amine groups of the doped PEI further enhanced that to 127.3%; meanwhile, doping low-molecular-weight PEI into PNP, while simultaneously doping high-molecular-weight PAA, avoided the precipitation of PAA and PEI, due to interaction of the two oppositely charged polymers; and these also made the self-assembly procedure more effective and the nanoparticle structure more stable than PNP and also led to the production of rich residual PAA chains on the surface of dPNP. The storage results showed that the average hydrated particle diameter kept almost constant (197.5−213.1 nm) during 15-day storage and that the nanoparticles have rich surface charge of −11.47 mV (zeta potential), well suspension stability and good dispersity without detectable aggregation in the solution during the storage. Therefore, the nanoparticle is quite suitable for the antibody labeling, immunoassay and the storage. As a result, a high-sensitive ECL immunoassay approach with good precision, accuracy and selectivity was established and an ultra-low detection limit of 0.049 pg mL−1 (S/N = 3) for magnetic bead-based detection of Hepatitis B surface antigen was observed.