Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.