Objective To investigate the effect of preoperative carbohydrate antigen 19-9 (CA19-9) levels on the prognosis of hepatocellular carcinoma (HCC) patients with different α-fetoprotein (AFP) levels. Methods The medical records and follow-up data of 3 791 HCC patients undergoing hepatectomy in our hospital from Jan. 4, 2008 to Dec. 31, 2010 were prospectively collected. When 400 ng/mL was taken as the cut-off value of preoperative AFP level and 32 U/mL as the cut-off value of preoperative CA19-9 level, the patients were divided into four groups:double positive group (DP group), CA19-9 single positive group[SP (CA19-9) group], AFP single positive group[SP (AFP) group] and double negative group (DN group). The tumor characteristics of the patients in the four groups were compared. Kaplan-Meier analysis and log-rank test were used to analyze the overall survival (OS) and disease-free survival (DFS) of each group. Univariate and multivariate analyses were performed using Cox proportional hazards model to screen the independent factors influencing the prognosis of HCC patients. Results Patients in the four groups had different tumor characteristics. Compared with the DN group, the tumor maximal diameters of patients in the SP (AFP) and DP groups were significantly larger, the percentages of patients with Edmondson-Steiner grade Ⅲ-Ⅳ were significantly higher and the positive rates of microvascular invasion (MVI) were significantly higher (P<0.01), and the proportion of multiple tumor in the DP group was significantly higher (P<0.05); while the tumor maximal diameter in the SP (CA19-9) group was significantly smaller (P<0.05), and the proportion of multiple tumor was significantly higher (P<0.01). The 1-, 3- and 5-year OS rates of patients in the DN group, SP (CA19-9) group, SP (AFP) group and DP group were decreased successively (P<0.01). The 1-, 3- and 5-year DFS rates of patients in the DN group were the highest (P<0.01), while those in the DP group were the lowest (P<0.01); there were no significant differences in the 1-, 3- or 5-year DFS rates between the SP (CA19-9) and SP (AFP) groups. The stratified analysis of preoperative AFP levels showed that the 1-, 3- and 5-year OS rates and DFS rates in the CA19-9<32 U/mL group were significantly higher than those in the CA19-9 ≥ 32 U/mL group. Multivariable analysis showed that AFP ≥ 400 ng/mL, CA19-9 ≥ 32 U/mL, intraoperative bleeding ≥ 600 mL, tumor maximal diameter ≥ 5 cm, multiple tumor, absence of tumor capsule, MVI, and Edmondson-Steiner grade Ⅲ-Ⅳ were independent risk factors of OS (P<0.05); hepatitis B surface antigen (+), AFP ≥ 400 ng/mL, CA19-9 ≥ 32 U/mL, tumor maximal diameter ≥ 5 cm, multiple tumor, absence of tumor capsule, and MVI were independent risk factors of DFS (P<0.05). Conclusion Preoperative serum AFP ≥ 400 ng/mL and CA19-9 ≥ 32 U/mL are independent risk factors of OS and DFS in HCC patients. Preoperative CA19-9 level is an important indicator to further assess the prognosis of HCC patients with different AFP levels.