The self-luminescence behavior of lanthanide MOFs (Ln-MOFs) due to the unique antenna effect is considered to be a promising electrochemiluminescence (ECL) emission for biosensors. It is more challenging for Ln-MOFs on account of the difficulty to stimulate Ln ions with the desired energy-transfer efficiency to produce stronger ECL emissions at a low potential. Here, guided by a second ligand-assisted energy-transfer strategy, we present an efficient self-enhanced luminescence mixed-ligand Eu-MOF as an ECL signal probe for an oriented antibody-decorated biosensing platform with a low detection limit and a broad detection range. Diamino terephthalic acid (NH2-H2BDC) and 1,10-phenanthroline (Phen) were selected as the first and second ligands, respectively, to form highly conjugated structures, as well as suppress the nonradiative energy transfer. Impressively, Phen precisely adjusts the energy gap between the triplet ligand and the excited state of Eu3+, realizing the self-enhancement of ECL efficiency of the Eu-MOF. The mixed ligand adjusted the molar ratio to obtain the stable and strong ECL signal at a lowered triggering potential (0.83 V). In addition, FeCo@CNT features densely active FeCo sites along with a rich hierarchy conductive carbon nanotube (CNT) network, which is efficiently a co-reaction accelerator to produce more TPA•+ radicals to accelerate the reduction process of the Eu-MOF for achieving the ECL emission amplification. FeCo@CNT with heptapeptide HWRGWVC (HWR) constructed a matrix biosensing interface that allowed the fragment antigen-binding (Fab) regions to target specific antigens and enhance the incubation efficiency. The present study has gone some way toward designing a self-enhanced luminous Eu-MOF, thus giving new fresh impetus to develop high-performance ECL emitters for biological analysis.