Introduction The intestinal mucosa of children and adult with coeliac disease shows characteristic changes in intraepithelial lymphocyte (IEL) populations. Objectives Determination of the normal range of IEL populations by flow cytometry and its diagnostic usefulness in coeliac disease (CD). Methods A retrospective study of 246 children and 461 adults with suspected CD with IEL immunophenotype results. The CD group (221 children and 98 adults) are individuals with positive coeliac serology and a histology lesion Marsh grade 1 or greater. The control group included 25 children and 363 adults without bowel lesion, negative serology and with IEL immunophenotype results. Results The group of coeliac patients, adults and children, shows a significant increase in total IEL and TCRγδ IEL, and a significant decrease in NK-like IEL compared with control groups. Based on ROC curves, the cut-off in coeliac children was: %IEL >14.2%, %TCRδγ IEL>16.5% and %NK-like IEL<10.1%. The cut-off in the adult coeliac population was: %IEL >14.2%, %TCRδγ IEL>16.1% and %NK-like IEL<4.4%. In both populations the specificity and PPV are close or equal to 100%, a CP+ >5 and a CP− <2 or near. Conclusions The cut-off values of the LIE population analysed has been established in this study. The values of CP+ and CP− show that these parameters are very useful for the diagnosis of celiac disease in children and adults, with a specificity of approximately 100%. The immunophenotyping of LIE is a very useful technique in the diagnosis of CD, and complements the classical pathological study, thus increasing the specificity.