This study was performed to estimate the prevalence of Enterotoxigenic Escherichia coli (ETEC) in calves' diarrhoea samples by molecular and serological methods. Rectal swabs from 127 diarrhoeaic calves were collected from 10 different farms of Tehran province during March to September 2014. ETEC was detected by both mPCR and ELISA. According to the results, ETEC was detected in 14 (11%) and 5 (3.9%) diarrhoea samples by mPCR and ELISA methods respectively. All 14 positive samples were detected by mPCR method had three virulence genes including K99 (F5), F41 and STa. Although 11 (78.5%) and 3 (21.4%) positive cases were identified from traditional and semi-industrial farms respectively, no isolates were identified related to the industrial farms. The results represented that detection assays such as ELISA which is only able to detect fimbriae (F5), will follow false-positive cases. For this reason mPCR assay has been developed and recognized as effective, rapid and reliable tools for detection of ETEC.