Background Giardia lamblia is a very common cause of gastrointestinal symptoms worldwide. There are several methods for the diagnosis of Giardia infection, however none are ideal. We aim to find a new, microRNA-based method that will improve the currently available diagnostic methods for giardiasis. Methods Deep-sequence profiling of Giardia small-RNA revealed that miR5 and miR6 are highly expressed in Giardia. These miRNAs were tested by qRT-PCR in duodenal biopsies of patients with giardiasis who were positive by microscopic pathological evaluation. The gastric biopsies of the same patients served as negative control tissues. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. Results All histologically proven duodenal biopsies of patients with Giardia infection were positive for Giardia miR5, with a mean threshold cycle (Ct) of 23.7, as well as for Giardia DNA qPCR (16S-like gene, mean Ct 26.3). Gastric biopsies which were tested as a control all were negative. Stool evaluation of miR6 in patients with giardiasis showed 90% specificity but only 66% sensitivity, and a lower accuracy rate was obtained with miR5. Conclusion Giardia miR5 testing in duodenal biopsies may be a new method for the diagnosis of giardiasis. It seems to be more sensitive when compared with testing for Giardia DNA by qPCR in duodenal biopsies. It will be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies from patients with persistent abdominal symptoms.