Despite considerable efforts, the fabrication of protein chips using a self-assembled monolayer (SAM) with a long chain length remains a challenge, due to its steric hindrance and disulfide deposited, which can generate multilayers and block the functional groups of the SAMs, and thus, significantly reduce the sensitivity of the chips. To improve their sensitivity, the feasibility of using a short-chain SAM, 4,4- Dithiodibutyric acid (4,4- DTBA), as a monolayer for the protein chips based on a gold surface is studied. Experiments for characterizing 4,4- DTBA are performed by atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and ellipsometry. Additionally, the fluorescent assay of 4,4- DTBA is characterized using protein A-FITC (fluorescein isothio cyanate) to investigate its binding capacity. The results of 4,4- DTBA are compared with those of 11- MUA (11-Mercapto-undecanoic acid). The comparison results indicate that 4,4- DTBA can be adopted as a monolayer for the protein chips.