Gold nanoparticle-catalyzed chemiluminescence (CL) of luminol is an attractive alternative to strategies relying on enzymes, as their aggregation leads to significantly enhanced CL signals. Consequently, analytes disturbing such aggregation will lead to an easy-to-quantify weakening of the signal. Based on this concept, a homogeneous aptamer-based assay for the detection of sulfadimethoxine (SDM) has been developed as a microfluidic CL flow-injection platform. Here, the efficient mixing of gold nanoparticles, aptamers, and analyte in short channel distances is of utmost importance, and two-dimensional (2D) and three-dimensional (3D) mixer designs made via Xurography were investigated. In the end, since 2D designs could not provide sufficient mixing, a laminated 3D 5-layer microfluidic mixer was developed and optimized with respect to mixing capability and observation by the charge-coupled device (CCD) camera. Furthermore, the performance of standard luminol and its more hydrophilic derivative m-carboxy luminol was studied identifying the hydrophilic derivative to provide tenfold more signal enhancement and reliable results. Finally, the novel detection platform was used for the specific detection of SDM via its aptamer and yielded a stunning dynamic range over 5 orders of magnitude (0.01-1000 ng/ml) and a limit of detection of 4 pg/ml. This new detection concept not only outperforms other methods for SDM detection, but can be suggested as a new flow-injection strategy for aptamer-based rapid and cost-efficient analysis in environmental monitoring and food safety.