An automated microfluidic photonic crystal enhanced fluorescence (PCEF) assay platform was recently reported for the multiplexed detection of cytokines spiked into phosphate buffered saline. In this work, we apply the aforementioned platform for cancer biomarker detection in human serum. Detection and control proteins were printed in a microarray format onto the surface of a photonic crystal designed to enhance the signal of fluorophores in the excitation and emission range of Cyanine 5 dye and similar alternatives. Fluorescence-linked immunosorbent assays (FLISAs) were performed, and a dose-response curve was obtained for human serum spiked with anti-E7 antibody, a known biomarker for human papillomavirus (HPV)-associated oropharyngeal cancer (OPC). Further, clinical samples of 20 OPC patients and 20 healthy controls were also tested using the PCEF platform, and the results were compared to those of the gold standard enzyme-linked immunosorbent assay (ELISA). Based on the results of a single trial of forty clinical samples, the sensitivity and specificity of the automated PCEF assay for anti-E7 is 75% and 65%, respectively, compared to 56% and 95% for ELISA.