Au−Ag alloy nanoparticles with a homogeneous size distribution and good stability were synthesized by simultaneous one-step reduction of HAuCl4 and AgNO3, characterized by transmission electron microscopy (TEM), energy dispersion X-ray spectrometry (EDS), scanning electron microscopy (SEM), and selected-area electron diffraction (SAED). The oxidation reaction between cerium(IV) and rhodamine 6G (Rho 6G) in a sulfuric acid medium brought weak chemiluminescence (CL). It was found that Au−Ag alloy nanoparticles could enhance the chemiluminescence of the rhodamine 6G−cerium(IV) system, and the most intensive CL signals were obtained by Au−Ag alloy nanoparticles with a molar ratio of 3:4. On the basis of the studies of UV−visible, CL, and PL spectra, the CL enhancement mechanism had been proposed; namely, rhodamine 6G and Au−Ag alloy nanoparticles were oxidized by cerium(IV) to form the excited-state cerium(III)*, which transferred energy to rhodamine 6G and the oxidized product of rhodamine 6G, resulting in light emission. Moreover, the influences of 17 amino acids, such as cystine, methionine, and phenylalanine, and 22 organics, including ascorbic acid, 2,4-dihydroxybenzoic acid, and p-aminobenzoic acid, on the rhodamine 6G−cerium(IV)−Au−Ag alloy nanoparticle CL system were studied by a flow-injection procedure, which led to an effective method to detect these compounds.