Although previous animal studies have indicated that certain micro ribonucleic acids (microRNAs) play a part in the pathway of opioid addiction, whether such findings extend to human models is yet unknown. This study aims to investigate the important microRNA expressions in patients with opioid use disorder (OUD) on methadone maintenance treatment (MMT) compared to healthy controls and analyze the correlation between microRNAs and opioid characteristics among the patients. We recruited 50 patients and 25 controls, and both groups were matched regarding gender, age, and body mass index. Serum microRNAs (miR-133b, miR-23b, miR-190, miR-206, miR-210, and miR-21) were measured. The age of OUD onset, duration of MMT participation, and recent daily methadone dosage were considered the opioid characteristics. We adopted the t-test to compare the difference between patients and controls and Pearson's correlation to evaluate the association between microRNAs and opioid profiles. Only the level of miR-133b in OUD patients on MMT was significantly lower than that in healthy controls. We did not detect differences of any other microRNA expressions between the two groups. Furthermore, we found no evidence to support the association between microRNAs and opioid characteristics. This study indicates that miR-133b values may be decreased in OUD patients on MMT.