Protein microarrays have shown a powerful tool to identify cancer biomarkers due to the multiplex detection and tiny sample requirement. However, there is no single biomarker that is sensitive and specific enough to meet strict diagnostic criteria due to the diversity and variation of cancers. To this end, a combination of biomarkers is required to increase sensitivity and specificity. In this book, various silane monolayers and polymer layers were developed to functionalize 3D micro-structured glass slides to effectively immobilize proteins, and the detection parameters were optimized for sensitive biomarker detection. Under the optimized conditions, protein microarrays reach a wide dynamic range and low limit of detection to pM range, but the analytical performance of each biomarker significantly depends on surface chemistry. The implemented protein microarrays were used for the detection of biomarkers from colorectal and breast cancer sera. Results show that the combination ofcancer biomarkers on their specific surface lead to remarkably increase of the positive responses. A panel of 5 biomarkers allows to specifically detect over 82% of breast cancer patients.