The diagnostic value of combining glypican-3 (GPC3) and CD34 staining for small nodules in liver biopsy specimens has not been evaluated. In this study, 201 thin-core biopsy specimens were assessed using GPC3 and CD34 immunochemical staining, including 33 cirrhotic regenerative nodules, 31 high-grade dysplastic nodules, 70 hepatocellular carcinomas (HCCs) with nodules 3 cm or smaller, and 67 HCCs with nodules larger than 3 cm. The results showed that the accuracy of GPC3 staining (90.3%) among liver nodules 3 cm or smaller was better than its use among all nodules (P = .045). Furthermore, the positive expression rate of costaining was significantly greater than that observed for GPC3 or CD34 single staining (P < .001 and P = .002, respectively). These data demonstrate that GPC3 staining is more accurate for the diagnosis of HCC on thin-core biopsy specimens in nodules 3 cm or smaller compared with its use in all nodules, while GPC3 and CD34 costaining has better diagnostic value than does single staining.