Staphylococcus  aureus  is  one  of  the  most  important  pathogens  in  hospital acquired infections. Annually, many people are infected with S. aureus in hospitals. Rapid detection of this bacterium is extremely helpful in preventing and managing this bacterium mediated diseases. Aptamers are powerful probes, which can be used as a target explorer in a wide range of diagnostic systems. To isolate a specific aptamer against S.  aureus,  a  library  of  single-stranded  DNA  molecules  was designed,  and  enriched  through  Cell-SELEX  procedure.  In the Cell-SELEX, the DNA library was exposed to the S.  aureus bacterium in 8 reiterative quadruple rounds including: binding, separation, elution and amplification. After 8 rounds, the PCR product was cloned and sequenced. Cloned aptameric sequences were evaluated through enzyme-linked oligonucleotide assay (ELONA), and a sequence with the best outcomes was selected as ideal aptamer. Eight rounds of Cell-SELEX procedure led to isolation of a specific ssDNA aptamer against S. aureus and named as “STAPT” (conflation of STAphylococcus and APTamer). Using ELONA technique, the detection limit of this aptamer was determined as 4 × 10³ CFU/ml. The aptamer “STAPT” showed the promising and potent abilities and features to be utilized as a bio-detection element likely in advanced detection systems. Although more extended researches are needed for this purpose.