An indirect analysis strategy for small organic molecules is presented herein, combining aptamer-modified gold nanoparticles (AuNPs) with a designed barcode sequence for amplification. Ethanolamine, which could not be analyzed directly through liquid chromatography coupled with mass spectrometry (LC/MS), was used as a model compound to demonstrate the feasibility of the proposed scheme. A poly-adenosine barcode sequence was part of the designed cDNA A1 (cDNA + barcode sequence) to provide a large number of adenine bases for signal amplification. In the presence of fixed amounts of the aptamer and cDNA A1, the concentration of ethanolamine could be estimated indirectly through measurement of the concentration of the hydrolyzed bases of the unbound cDNA A1. The thiol-terminated aptamer was immobilized on the AuNPs through Au–S self-assembly. The aptamer-immobilized AuNPs were removed, with their bound ethanolamine and cDNA A1, through centrifugation. The unbound cDNA A1 remaining in the supernatant was hydrolyzed and analyzed. Through indirect analysis and amplification of the cDNA A1, the concentration of ethanolamine could be analyzed with a linear range (on a logarithmic scale) between 5 and 5000 nM (detection limit: 1.2 nM). This developed method might be applied as a general platform for indirect detection using LC/MS analysis.