Objective To explore the expression of CD34 and polyclone carcinoembryonic antigen (pCEA) of positive and negative alpha fetoprotein (AFP) detected by puncture biopsy in human hepatocellular carcinoma (HCC) and the significance of pathological diagnosis. Methods Fifty-four HCC tissue specimens from 2013 to 2015 were collected from tumor biopsy samples which confirmed by pathology in the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture. The tissue samples were divided into positive AFP group (n=33) and negative AFP group (n=21) according to the detection results of serology and immunohistochemistry analysis of AFP. Expressions of CD34 and pCEA in the fifty-four HCC specimens were detected by immunohistochemistry. Results The positvie expression rate of pCEA in the positive AFP group was 69.7%, which was significantly higher than that in the negative AFP group (38.1%) (P<0. 05). However, the difference in positive expression rate of CD34 between the positive and negative AFP groups (90.91% and 85.71%, respectively) was not significant (P>0.05). Conclusion The associated detection of AFP, pCEA and CD34 in HCC tissues might contribute to the pathological and differential diagnosis of human hepatocellular carcinoma in puncture biopsies.