The use of a low-risk and convenient diagnostic method is important in the early diagnosis of achalasia and could prevent the side effect of delay in initiation of treatment. According to our previous NGS study, two miRNAs, hsa-miR-217 and hsa-miR-143-3p were differentially expressed in achalasia esophageal tissue. The present study aimed to assess two microRNAs, hsa-miR-217 and hsa-miR-143-3p as serum biomarkers for early detection of achalasia. Serum was collected from patients with achalasia and non-achalasia participants. The relative expression of hsa-miR-217 and hsa-miR-143-3p were determined using quantitative real-time polymerase chain reaction. The expression of hsa-miR-217 was different in the serum of patients with achalasia and controls, but this difference was not significant (p-value = 0.572). Besides, the results showed that the serum expression of miRNA-143-3p was not significantly different between the patients with achalasia and the control group (p-value = 0.366). Our findings could not support serum miRNA-217 and miRNA-143-3p as potential diagnostic biomarkers for achalasia.