Objective: To investigate the diagnostic value of the combination detection of the glypican 3 (GPC-3) mRNA expression in peripheral blood monouclear cells (PBMC) and plasma level of γ-glutamyl transpeptidase (GGT) for patients with α-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). Methods: The PBMC GPC-3 mRNA expressions in 39 patients with AFP-negative HCC, 39 patients with non-HCC liver diseases and 40 healthy volunteers were determined by RT-PCR method, and the plasma GGT levels (>58 U/L was considered as positive) in the 39 AFP-negative HCC cases were measured by using automatic biochemical analyzer. The positive rates by single or combination of the two detections were analyzed. Results: The detection rate of PBMC GPC-3 mRNA expression in the 39 AFP-negative HCC patients was 62% (24/39), while none of the cases in patients with non-HCC liver diseases and healthy subjects presented positive expression of PBMC GPC-3 mRNA, thus the specificity of PBMC GPC-3 mRNA for AFP-negative HCC was 100%. The positive rate of GGT elevation in the 39 AFP-negative HCC patients was 80% (31/39). The positive rate of the combination detection of PBMC GPC-3 mRNA and GGT reached 95%, which was significantly higher than that of either GPC-3 mRNA or GGT alone (both P<0.05). Conclusion: PBMC GPC-3 mRNA expression has a high specificity in AFP-negative HCC patients. The combination detection of GPC-3 mRNA and GGT is helpful to the early diagnosis of AFP-negative HCC.