Objective To discover potential serum biomarkers of osteosarcoma by an integrated analysis of datasets from gene chip and surface enhanced laser desorption-ionization time-off-light mass spectrometry (SELDI-TOF-MS).Methods Gene chip was used to screen potential biomarkers of osteosarcoma from osteosarcoma cell lines (MG-63,Saos-2 and U-2OS),and SELDI-TOF-MS was used to screen protein peaks with statistically significant difference from serum samples (27 osteosarcoma cases and 47 healthy cases).The association between these two types of markers was constructed by link-test to get cross-validated serum biomarkers of osteosarcoma.Immunoblot analysis was performed to detect the expression of one candidate osteosarcoma protein maker in serums from osteosarcoma patients and members of the control group.Results Six hundred and fifty three candidate osteosarcoma gene markers were found by gene chip and 6 protein peaks were found by SELDI-TOF-MS.Thirteen differentially expressed serum markers of osteosarcoma including CYC-1 were screened by link-test analysis (12 of them were up-regulated in serum samples of the osteosarcoma patients while 1 was down-regulated ).By immunoblot,we found that CYC-1 protein was significantly up-regulated in serums from osteosarcoma patients compared to the control group.Conclusion An integrated analysis of datasets from gene chip and SELDI-TOF-MS could accelerate the discovery,screening and identification of tumor biomarkers.A total of 13 potential serum protein markers of osteosarcoma were discovered in this study.