A DNA aptazyme consists of an aptamer domain and a DNAzyme module, in which the DNAzyme activity can be regulated by the aptamer-target interaction. The complex of G-quadruplex (GQ) and hemin is a peroxidase-mimicking DNAzyme and has become increasingly popular as a reporter system for biosensing applications. The development of GQ-based aptazymes is of high interest as they can be used as label-free biosensors for the real-time detection of pathogens. Herein, we rationally designed ca. 200 GQ-based aptazyme candidates and evaluated the suitability of 14 aptamers targeting quinine, Protein A, Staphylococcus enterotoxin B, and ATP for this detection concept. As a result, six novel aptazymes were developed for the specific detection of quinine based on two quinine-binding aptamers. The rest of designed probes, however, hardly showed significant functionality. To uncover the reasons, we performed enzyme-linked oligonucleotide assays to find how the affinity of aptamers is affected once conjugated to the DNAzyme sequence or upon integration into the aptazyme probe. Furthermore, we investigated the impact of the structure-switching functionality in the parent aptamer and the effect of the reaction matrix on the efficiency of probes.