Free radicals play important roles in many physiological and pathological pathways in biological systems. These free radicals can be detected and quantified by their EPR spectra. The measured EPR spectra are often mixtures of pure spectra of several different free radicals and other chemicals. Blind source separation can be applied to estimate the pure spectra of interested free radicals. However, since the pure EPR spectra are often not independent of each other, the approach based on independent component analysis (ICA) cannot accurately extract the required spectra. In this paper, a novel sparse component analysis method for blind source separation, which exploits the sparsity of the EPR spectra, is presented to reliably extract the pure source spectra from their mixtures with high accuracy. This method has been applied to the analysis of EPR spectra of superoxide, hydroxyl, and nitric oxide free radicals, for both simulated data and real world ex vivo experiment. Compared to the traditional self-modeling method and our previous ICA-based blind source separation method, the proposed sparse component analysis approach gives much better results and can give perfect separation for mixtures of superoxide spectrum and hydroxyl spectrum in the ideal noise-free case. This method can also be used in other similar applications of quantitative spectroscopy analysis.