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A novel sandwich-type electrochemiluminescence (ECL) immunosensor was successfully fabricated for the ultrasensitive detection of avian leukosis virus subgroup J (ALV-J). The immunosensor used a glassy carbon electrode decorated with gold nanoparticles (Au NPs), primary antibodies (Ab1) and bovine serum albumin as the sensor platform. For the signal label, three-dimensional flower-like MnO2 clusters modified with polyethyleneimine and palladium nanoparticles (MnO2–PEI–Pd nanocomposite) were used as the novel carrier of Ru(bpy)3Cl2 and secondary antibodies (Ab2). Under optimal conditions, the ECL immunosensor offered a linear relationship between the ECL signal and the logarithm of the ALV-J concentration in the range from 101.98 to 104.30 TCID50/mL with a detection limit of 101.90 TCID50/mL (based upon a signal-to-noise ratio of 3). In addition, the immunosensor showed excellent stability, selectivity and reproducibility, thus offering an effective tool for the quantitative detection of ALV-J in clinical applications. [ABSTRACT FROM AUTHOR]