Background and Objectives: Staphylococcal enterotoxin D as a supper antigen is produced by infected samples of human and animal sources. The aim of this study was to standardize the detection methods for the Staphylococcus strain producing enterotoxin D. Materials and Methods: A PCR method was set up for detection of enterotoxin D gene (ent D) in Staphylococcus aureus samplesisolated from the human subjects (310 strains isolated from clinical samples). The specific PCR-product (a band about 700 bp) was purified and sent off for DNA sequencing. Blast analysis showed a 99% identity with the standard gene sequence from Genebank. The ability to produce enterotoxin D by all strains carrying ent D was analyzed by using an ELISA kit. Results: The results of this study show that the PCR method has been well set up. There were two PCR products obtained by the primer pair, one at 700 bp and another at 1400 bp. Both bands were gel purified and sent for DNA sequencing. The results, based on the alignment with the standard ent D sequences from GenBank, suggest that ent D is contained within the 700-bp product. Production of the entrotoxin D in the positive strains was confirmed by ELISA. Conclusion: Based on the available information, coagulase positive Staphylococcus aureus strains are recorded in clinical samples. However, there is no routine method available to analyze the ability of the bacterial strains for producingtoxins including enterotoxin D. This study represents a simple, fast, and standard method for verification of the bacteria enterotoxin D and the strains producing it.