For oral cancer, numerous saliva- and plasma-derived protein biomarker candidates have been discovered and/or verified; however, it is unclear about the behavior of these biomarker candidates as saliva or plasma biomarkers. In this study, we developed two targeted, multiplexed quantification assays, MRM and SISCAPA-MRM, to quantify 30 potential protein biomarkers in both plasma and saliva samples collected from 30 healthy controls and 30 oral cancer patients. In comparison with MRM assay, SISCAPA-MRM applying the anti-peptide antibodies for an additional immuno-enrichment effectively improved (>1.5 fold) the detection sensitivity of 11 and 21 targets in measurement of saliva and plasma samples, respectively. The integrated results revealed that the salivary levels of these 30 selected biomarkers weakly correlated (r < 0.2) to their plasma levels. Five candidate biomarkers (MMP1, MMP3, TNC, CSTA and PADI1) exhibited significant alternations and disease-discriminating powers (AUC >0.7) in saliva sample; nevertheless, no such targets could be found in plasma samples. Our data support the notion that saliva may be more suitable for the protein biomarker-based detection of oral cancer, and the newly developed SISCAPA-MRM assay could be applied to further verify the clinical utility of multiple oral cancer biomarker candidates in large cohort of saliva samples.