This study was designed to develop novel and better reliable serum prognostic biomarkers for colorectal cancer (CRC). A 50 sample set including CRC, adenoma and healthy control sera was used to identify the serum proteins involved in CRC carcinogenesis using serum proteomic approach. Alpha-2-glycoprotein 1, zinc-binding (AZGP1), pigment epithelium derived factor (PEDF) and peroxiredoxin 2 (PRDX2) were selected as good candidates. Two independent cohorts of 868 individuals were enrolled. The expression of selected proteins in serum from cohort 1 (n = 534) was quantified with enzyme-linked immunosorbent assays. CRC sera of this cohort (n = 405) were assigned to training and test sets, which were used to identify and verify the prognostic markers. The prognostic values of identified proteins were further validated in cohort 2 (n = 334) using quantitative reverse transcription PCR and immunohistochemical staining. Our data showed that the elevated AZGP1 and decreased PEDF and PRDX2 expressions in CRC serum and tissues were correlated with liver metastases. In the training set, higher AZGP1 and lower PEDF levels in sera were significantly associated with a poorer overall survival (OS), higher AZGP1 was also associated with a poorer disease-free survival (DFS). This association was verified in the testing set and further validated in patients in cohort 2. Patients with lower PEDF or PRDX2 levels in their CRC tissues had a significantly poorer DFS or OS than patients with high levels of these proteins in cohort 2. Univariate and multivariate analyses indicated that the prognostic performance of serum AZGP1 and PEDF was independent of other clinicopathological factors. We propose that they may serve as prognostic markers and potential therapeutic targets in CRC.