The time varying EEG spectral analysis is proposed as an objective approach to evaluate fatigue during cognitive process in SSVEP-based BCIs. The proposed approach can provide a real-time evaluation of the fatigue with objective and quantitative measurement. The promising result suggests the potential of objective evaluation of fatigue by dynamic measurement in EEG indices θ and α, as well as ratio indices θ/α, (θ + α)/β. The time varying EEG activity would predict the fatigue immediately, which may provide a useful online assessment of fatigue. The increase in θ, α and (θ + α)/β, as well as the decrease in θ/α are associated with the increasing fatigue level during cognitive process. In addition, this method can be used for optimal selection of visual stimuli parameters (e.g., visual stimulus frequency, duty cycle, color, etc.) to design a user-friendly BCI system, which cause less fatigue in further study.