More than 20 types of S. aureus enterotoxins have been identified so far. Detection each of them is very important in clinical samples. Different detection primers pair have been designed and used. The aim of this study was to assay the efficacy of three pair primers to detect staphylococcal enterotoxin type C in synovial fluid of RF patients and compare the results with Elisa. In this study, seventy synovial fluids of RF patients were assayed. Three pair primers were used. The primers were amplified 102, 206 and 1223bp fragment respectively. The PCR products were sequenced and compared multiple alignments with reference gene. In addition, the Elisa plate was designed for detection of the SEC. The obtained data was subjected to descriptive analysis. The results showed that the three pair primers were amplified by different frequencies. The results of amplified of amplicons 1223, 206 and 102bp were 9(18%), 34(68%) and 17(44%) positive for ent C gene respectively. The results of Elisa revealed that 22 cases (44%) were positive for staphylococcal enterotoxin C. The amplicon 206bp were the most abundant probability of the amplification product. The inter gene amplicons amplification pair primers were shown more specify. The results indicated that the staphylococcal enterotoxin C gene existed in synovial fluids of RF patients. Therefore candidate specific primers to amplify 206bp fragments of ent C and Elisa method are reliable. This finding may help to detect the etiology of rheumatoid arthritis.