A wide diversity of serological biomarkers is available for clinic diagnosis but individually their lack of specificity decreases their diagnostic and prognostic values. Protein microarrays have demonstrated to be powerful for screening and identifying biomarkers from minute samples. However, efficient multiplex analysis remains challenging due to biomarkers variability. A lot of parameters (surface chemistry, composition and pH of the spotting buffer, blocking reagents, protein concentration, etc.) have to be optimized for obtaining sensitive and specific detection. We developed 3D-microstructured chips for the implementation of customized antibody and antigen microarrays. These 3D-protein chips were evaluated for the sensitive detection, screening and profiling of biomarkers in various cancer sera.