Staphylococcus aureus bacteria is a ubiquitous Gram-positive microorganism that causes infections related to the sudden infant death syndrome. Recently, basic detection methods depend on complicated PCR amplification, electric separation, spectric adsorption and other detection systems. However, in this study, simplified sensitive voltammetric skills are developed. To identify an effective diagnostic method for Staphylococcus aureus (SA), a voltammetric sensing probe was sought using mercury immobilized on a carbon nanotube sensor (MCN). The voltammetric MCN conditions were optimized through stripping and cyclic voltammetry. Diagnostic electrolyte was used on non-treated blood sera as an electrolyte solution. The optimum cyclic and stripping analytical working range was 0.5–4.0 mL (3 × 102~5 × 102 CFU/0.5 mL) SA. The statistic relative standard deviation of 0.1 mL SA was observed to be 0.0078 (n = 5). Using the optimum parameters, a diagnostic test was performed by the direct assay of SA in non-treated human blood and patient sera. Here, the developed results can be used for the direct assay of non-treated blood sera, organ monitoring, in-vivo diagnosis, and other assays requiring SA detection.