A novel p16 protein (Pp16) electrochemiluminescence (ECL) biosensor using optical multi-metal nanocomposites as excellent nanocarriers was sucessfully constructed. The graphene oxide doped ferroferric oxide (GO@Fe3O4) was coated on the magnetic glassy carbon electrode (MGCE) via magnetism to form a functional magnetic electrode (GO@Fe3O4 MGCE). The GO@Fe3O4 MGCE used as supporting substrate for capture antibody (CAb), was significantly increased the amount of CAb and its stability. Meanwhile, ruthenium(II) tris-(bipyridine) (Ru)-doped gold-silver optical multi-metal nanocomposites (AgRuAu) were used as excellent nanocarriers for detecting antibody (AgRuAu@DAb). After immunological reaction, CAb-Pp16-AgRuAu@DAb was obtained and a strong ECL signal was synchronously generated. The change of the ECL signal showed a good linear relationship with the concentration of Pp16 and it ranged from 0.1 ng·L−1 to 0.1 μg·L−1. The first trace-level ECL magnetic-biosensor for Pp16 with the detection limit of 0.05 ng·L-1, exhibited outstanding analytical performances. Pp16 levels in normal human blood samples could be achieved with satisfying recovery values. The assay provides a promising tool for the analysis and evaluation of various tumor markers.