Compared with the well learned amplitude-frequency characteristic of Steady State Visual Evoked Potential (SSVEP), the effect of duty cycle is still unclear. In this work, the influence of duty cycle on SSVEP response is investigated in differnt frequency domains. The amplitude surface with the change of both frequencies and duty cycles is plotted. To get a stable response, the experiment arranged in 3 days, and each result is an average of 12 repetitions. It is interesting that the results from power spectral density (PSD) and canonical correlation analysis (CCA) method are not consistent. In addition, the relation between the fundamental component and its second harmonic component with the change of duty cycle is quite different at frequency of 7Hz, 10Hz and 13Hz. Based on the amplitude surface, we try to configure the subject-specific SSVEP based BCI. The frequencies and duty cycles of the stimulus are selected corresponding to the higher SSVEP response in the amplitude surface. Cross validation results show a significant improvement in the performance for the adjustment of duty cycle.