Background: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide. Less than 30% of patients are suitable candidates for surgery. Transarterial chemoembolization (TACE) is considered as a first-line treatment for patients with unresectable advanced liver cancer. In the routine diagnosis and treatment pathway for unresectable HCC, a biopsy is usually performed firstly, followed by hepatic artery angiography and TACE. However, hepatic artery angiography data reveals that the risk of arteriovenous shunt (AVS) is significantly increased following biopsy, which negatively affects the outcomes of TACE. Aim: To investigate the feasibility of delayed biopsy following TACE in patients with HCC. Methods: Data from 112 patients with a definitive diagnosis of HCC were retrospectively analyzed. Patients who underwent biopsy immediately after TACE formed the experimental group (n = 55) and those who underwent biopsy before TACE formed the control group (n = 57). Positive pathological diagnosis rate, incidence of AVS, and rates of TACE-related complications were compared between the two groups. In addition, factors affecting the occurrence of AVS were assessed. Results: There was no significant difference in positive pathological diagnosis rate between the experimental and control groups (81.8% vs. 77.2%, respectively; P = 0.545). The incidence of AVS in the experimental group was lower than that in the control group (3.6% vs. 22.8%, respectively; P = 0.003), and embolization results were better in the experimental group. There was no difference in the incidence of TACE-related complications between the two groups. Late tumor stage (P = 0.04) and pre-TACE biopsy puncture (P = 0.003) are associated with the occurrence of AVS. Conclusion: In patients with HCC, delayed biopsy following completion of TACE did not affect pathological diagnosis results and yielded better embolization outcomes. Therefore, delayed biopsy following completion of TACE in patients with HCC is worth popularizing for clinical use. However, with the development of precision medicine, the diagnosis and treatment of tumor will certainly advance to the molecular level, whether the ischemic and oxygen-deficient tumor microenvironment caused by TACE treatment will have an impact on tumor tissue at molecular level remains unknown.