This study was designed to examine the association between Blastocystis hominis and irritable bowel syndrome(IBS). In this case-control study that conducted in Mashhad, Iran in 2014-2015, one hundred IBS cases and one hundred matched (age and sex) healthy people were participated. Direct stool examination, formalin-ether concentration technique and trichrome staining were done. The data were analyzed by SPSS20 with Fisher's exact test and T-test. One hundred IBS patients (31 males and 69 females) had a mean age of 29.5 (±7.4) years. B. hominis were positive in 26% of IBS participants, and 9% in control group (P-value=0.002,Risk Estimate=3.5). Giardia lamblia were positive in 6% of IBS participants, and in none of control participants (P-value=0.01). Trichrome staining for detection of B. hominis was more sensitive than direct examination and formalin-ether concentration technique (P-value <0/001). B. hominis was more frequent in patients with irritable bowel syndrome. For detection of B. hominis, trichrome staining was more sensitive than other methods. Authors suggest that in patients with IBS, stool should be examined with different methods for three times to obtain a more reliable diagnosis.