Electrogenerated chemiluminescence (ECL) of a novel phenyleneethynylene derivatives [4,4’-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid; P-acid] possessing carboxylic acid groups at the para positions in aqueous solutions and its first immunosensing application are studied. Nanotubular mesoporous Pt–Ag alloy nanoparticles are first used to fabricate the ECL immunosensor as a signal amplification carrier. Absorption, photoluminescence, cyclic voltammetry, scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy are used as powerful tools to characterize P-acid, Pt–Ag alloy nanoparticles, and the fabrication process of the immunosensor. P-acid exhibits two ECL peaks at –1.07 (ECL-1) and 0.64 (ECL-2) V in air-saturated pH 7.4, 0.1 M PBS containing 0.1 M KCl during the cyclic sweep between 1.51 and –2.67 V at 0.1 V s−1; the ECL mechanism of these two ECL peaks is demonstrated. The electron-transfer reaction between electrochemically oxidized P-acid and tri-n-propylamine (TPrA) radical cation is also demonstrated to produce high ECL emissions and used to develop an ultrasensitive ECL immunosensor. In addition, a Pt–Ag alloy nanoparticles amplification carrier for protein ECL analysis is applied for improvement of the detection sensitivity. Thus, this ECL immunosensor exhibits high sensitivity, good reproducibility, rapid response, and long-term stability.