It is of great importance to efficiently immobilize probes onto a substrate with good spot quality for fabrication of protein microarrays. Printing buffers play an essential role in the fabrication process for the microarrays. In this work, antigen (Ag)/antibody (Ab) microarrays were fabricated on 3-aminopropyltriethoxysilane (APTES) modified glass slides through glutaraldehyde (GA), a bis-aldehyde homobifunctional cross-linker. Different types of buffers such as triton X-100 and glycerol and their effects on the protein immobilization were investigated for improving the quality of microspots and the immobilization efficiency on the aldehyde-activated APTES silanized slides. In addition, the performance of the optimized printing buffer was characterized with fabricated Ag/Ab microarrays. The results indicated that the optimized printing buffer, 0.01 M PBS with additional 0.003% triton X-100 and 10% glycerol could effectively eliminate non-homogeneous morphology of the microspots and significantly improve the signal intensities. The results provide an improved approach to construct high performance Ag/Ab microarrays.