Provided is luminescent gold nanomaterial functionalized by N-(4-aminobutyl)-N-ethylisoluminol, methods of preparation and application thereof. The functionalized gold nanomaterial is formed by N-(4-aminobutyl)-N-ethylisoluminol bonding to the surface of the gold nanoparticle. The functionalized gold nanomaterial are prepared by directly reducing chloroauric acid with N-(4-aminobutyl)-N-ethylisoluminol, wherein N-(4-aminobutyl)-N-ethylisoluminol acts as reducer and stabilizer simultaneously. The preparation method is simple, fast and no need of special conditions. The preparation methods can be performed in a wide temperature range, for example, 15-35.degree. C. The size and pattern of the functionalized gold nanomaterial can be specified by choosing the ratio of chloroauric acid to N-(4-aminobutyl)-N-ethylisoluminol. The obtained functionalized gold nanomaterial exhibits excellent chemiluminescence properties. Said functionalized gold nanomaterial can be combined with biomolecules to form biomolecular probe, which can be used for immunoassay, nucleic acid analysis, molecular imaging, sensor, etc., and has a broad application prospect in the field of clinical analysis biomedicine, food safety, and environment monitoring.