A stable and versatile biosensor surface is a desirable feature for any sensor, and the DNA-directed immobilization (DDI) of protein-DNA conjugates on the mixed ssDNA/ Oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) offer an alternative choice for preparing various types of protein chips. The patterned DNA chip is suitable for long-term storage and easily converted into a protein chip in one simple step. In our previous study, the experimental results indicated that a 50:1 ratio of OEG: COOH-terminated OEG and DNA sequences with 20 mer are the best conditions or making a protein chip via a DNA-directed immobilization (DDI) method. In this study, we investigate the effect of buffer pH values on the measurement of human serum albumin (HSA) by using the protein chip prepared by DDI method. The results showed that the largest SPR response is found in the detection of HSA with the use of 10 mM MES buffer (150 mM sodium chloride, adjusted to pH 4.0). We suggest that this was a consequence from the contribution of attractive electrostatic force between HSA and the negatively charged sensor surface. The lowest detection limitation of HSA with the use of MES buffer is 0.02 mg/ml. In addition, there is a linear relationship between the SPR signals and HSA concentrations (from 0.02 mg/ml to 0.5 mg/ml). Chip regeneration experiments were also performed in our study with the results showing that the average response for HSA detection on regenerated surface was nearly 92.3% of the response produced by using fresh surface.