Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with an increasing incidence. The exploration of biomarkers for HCC is one of the main aims for improving the efficacy of diagnosis and treatment. The microarray technology provides a high-throughput platform for parallel exploration of biomarkers for clinics. In this study, we used antibody microarrays to screen the novel cytokine biomarkers of hepatitis B virus (HBV)-related HCC. Cytokine-secreting patterns in sera were determined from 109 cases including 43 HBV-related HCC patients, 33 chronic hepatitis B patients, and 33 normal controls by RayBio Biotin label-based human antibody array. The correlation analysis was performed with conventional clinical diagnostic biomarkers, including serum alanine aminotransferase, alpha-fetoprotein (AFP) and hepatitis B surface antigen. Our results showed that in HBV-related HCC group, which had the highest percentage of AFP positive (>20 ng/ml) ratio, six cytokines were found differentially expressed in HCC patients (P < 0.05), compared with either normal controls or chronic hepatitis B group. Two macrophage-related cytokines, macrophage-derived chemokine (MDC) and macrophage-stimulating protein α (MSPα), displayed significant difference in the HCC group. Furthermore, an HCC diagnostic model for prediction was constructed, by which the combination of MDC and MSPα together with AFP had improved the diagnostic sensitivity from 60% (AFP alone) to 73.2% with similar specificity. Our results suggested that MDC and MSPα screened by antibody microarrays might serve as novel cytokines biomarkers for potential auxiliary diagnosis of HBV-related HCC.