Alpha-toxin of Staphylococcus aureus belongs to the pore-forming toxin (PFT) family, which can lyse red and white blood cells. In addition to the existence of the hla gene in the majority of S. aureus strains (about 95 %), higher expression exhibits enhanced pathogenicity to the bacteria. Various methods, such as antibodies and aptamers, could serve to detect this toxin. In the current study, for the first time, an improved sandwich aptamer-antibody-based method was developed using specific murine polyclonal antibodies and a specific aptamer to detect a wide range of α-toxin levels. Denatured recombinant α-toxin was administered to mice to trigger the production of specific antibodies, which were subsequently purified from immune sera. These antibodies served as capturers in the designed apta-qPCR assay, with an aptamer employed as a detector. The results showed that spiked α-toxin in the undiluted serum samples could detect α-toxin between 300 and 0.5 ng/mL with no cross-reactivity. The coefficient of variation (CV) percent of intra- and inter-assays were 0.84 and 1.06, respectively. Since in the apta-qPCR assay, a combination of specific polyclonal antibodies as capture and specific aptamer along with real-time PCR (qPCR) sensitivity is used, this robust method could be used in diagnostic laboratories to detect various levels of the toxin in human serum samples.