Background: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. New serum biomarkers for HCC screening are needed, especially for alpha-fetoprotein (AFP) negative patients. As a proximal fluid between body fluids and intracellular fluid, tissue interstitial fluid (TIF) is a suitable source for serum biomarker discovery. Methods: Sixteen paired TIF samples from HCC tumour and adjacent non-tumour tissues were analysed by isobaric tags for relative and absolute quantitation (iTRAQ) method. Two proteins were selected for ELISA validation in serum samples. Results: Totally, 3629 proteins were identified and 3357 proteins were quantified in TIF samples. Among them, 232 proteins were significantly upregulated in HCC-TIF and 257 proteins down-regulated. Two overexpressed extracellular matrix proteins, SPARC and thrombospondin-2 (THBS2) were selected for further validation. ELISA result showed that the serum levels of SPARC and THBS2 in HCC patients were both significantly higher than those in healthy controls. The combination of serum SPARC and THBS2 could distinguish HCC (AUC = 0.97, sensitivity = 86%, specificity = 100%) or AFP-negative HCC (AUC = 0.95, sensitivity = 91%, specificity = 93%) from healthy controls. And the combination of serum SPARC and THBS2 could also distinguish HCC patients from benign liver disease patients (AUC = 0.93, sensitivity = 80%, specificity = 94%). In addition, serum THBS2 was found to be a novel independent indicator for poor prognosis of HCC. Conclusions: Novel HCC candidate serum markers were found through in-depth proteomic analysis of TIF, which demonstrated the successful utility of TIF in cancer serum biomarker discovery.