Objective To evaluate the value of serum alpha-fetoprotein-L3 (AFP-L3) and hepatitis B virus DNA (HBV DNA) detection in diagnosis of hepatocellular carcinoma (HCC) secondary to HBV-related cirrhosis. Methods Serum samples were collected from 66 patients with HCC secondary to HBV-related cirrhosis and 71 cases with HBV-related cirrhosis. HBV DNA content was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). AFP-L3 was separated by micro centrifugal purification column. The levels of AFP-L3 and total AFP were quantified by electrochemiluminescence immunoassay; the percentage of AFP-L3 to total AFP was calculated. Statistical analysis was performed using SPSS 19.0 for Windows. Results The positive rates of AFP-L3 and HBV DNA in the patients with HCC were 74.2% and 59.1% respectively, but in the patients with cirrhosis they were 9.9% and 33.8% respectively. The positive rates of AFP-L3 and HBV DNA in the patients with HCC were both higher than those in the patients with cirrhosis (χ2 = 58.667 and 8.806, P = 0.000 and 0.003).The results of AFP-L3 and HBV DNA were consistent (Kappa = 0.748). In parallel diagnostic test, the sensitivity of combined detection was higher than that of AFP-L3 alone (χ2 = 6.371, P = 0.012). There was no significant difference between the specificity of combined detection and that of AFP-L3 alone in the diagnosis of HCC secondary to HBVrelated cirrhosis (χ2 = 0.132, P = 0.716). Conclusions The results of AFP-L3 and HBV DNA are consistent to some extent, they both can be regarded as important markers of HCC. The parallel test of combining AFP-L3 and HBV DNA can improve the sensitivity for diagnosis of HCC, can effectively reduce missed diagnosis of HCC.