A novel electrochemical immunosensor for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) was designed by using graphene sheets (GS)-layered double hydroxides (LDHs) composites modified electrode with multifunctional Fe3O4 core/Ni–Al LDHs shell (LDHs@Fe3O4) nanospheres as labels. At first, the GS-LDHs were used for the immunosensor platform for improving the electronic transmission rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab1). After that, ferrocene (Fc), secondary antibodies (Ab2) and horseradish peroxidase (HRP) multifunctional LDHs@Fe3O4 nanospheres were used as labels with high load amount and good biological activity. Subsequently, in presence of H2O2, amplified signals were obtained by an electrochemical sandwich immunoassay protocol. To embody the signal amplification property of the protocol, the analytical properties of various immunosensor platform and labels were compared in detail. Under optimal conditions, the reduction peak currents of the electrochemical immunosensor were proportional to the ALVs-J concentration over the range from 102.32 to 105.50 TCID50/mL with a low detection limit (180 TCID50/mL, S/N=3). The resulting immunosensor also displayed a good selectivity, reproducibility and stability.