Previous studies have identified a number of microRNAs (miRNAs) that were aberrantly expressed in hepatocellular carcinoma (HCC) tissues. Nevertheless, their diagnostic and prognostic value in serum has not been fully evaluated. Herein, the levels of five serum miRNAs, namely, miR-182, miR-331-3p, miR-197, miR-492, and miR-581, were detected in 103 HCC patients, 95 benign liver diseases, and 40 healthy controls using real-time PCR technique. The results showed that, compared with benign liver diseases and healthy controls, the levels of serum miR-182 and miR-331-3p were significantly increased in HCC patients, both P < 0.001. Area under the receiver-operating characteristic (ROC) curves for serum miR-182 and miR-331-3p were 0.911 (95 % CI, 0.863-0.947) and 0.890 (95 % CI, 0.838-0.930), the sensitivity were 78.6 and 79.61 %, and the specificity were 91.58 and 86.32 %, respectively. Moreover, the combination of serum miR-182, miR-331-3p, and alpha-fetoprotein (AFP) can markedly increase the differential diagnostic value of benign and malignant liver diseases, especially better than serum AFP alone, P < 0.05. Serum miR-182 was positively correlated with serum AFP (P = 0.001), tumor size (P = 0.013), and TNM classification of malignant tumors (TNM) stage (P = 0.003); however, only TNM stage was demonstrated a significant correlation with serum miR-331-3p (P = 0.006). In addition, Kaplan-Meier survival curve, together with univariate and multivariate Cox proportional hazard analyses, further disclosed that serum miR-182 and miR-331-3p were associated with postoperative survival of HCC patients, and both of them were regarded to be independent prognostic factors for patients with HCC. Taken together, our present study indicates that serum miR-182 and miR-331-3p, upregulated in HCC, can provide positive diagnostic and prognostic values for HCC.