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Electrochemiluminescence (ECL) of poly‐amidoamine (PAMAM) dendrimers functionalized to Fe3O4 nanoparticles (PAMAM@Fe3O4 NPs) based on Ru(bpy)32+ complex is reported. Different generations of PAMAM dendrimer consisted of 5 (D5), 10 (D10), 8 (D8) and 16 (D16) surface primary amine‐terminated groups. D5@Fe3O4 NPs enhanced ECL emission in comparison to others. A possible mechanism is elucidated based on structural functionality of PAMAM with respect to amines, carbonyl, hydroxyl groups and size‐based diffusion. ECL biosensing of alpha‐fetoprotein (AFP) and glypican‐3 (GPC‐3) using 1 mM tri‐propylamine is demonstrated using D5@Fe3O4NP@antibodies as detection probes. Detection limit for AFP and GPC‐3 was 6 pg/mL and 0.03 pg/mL, respectively.