The carboxylated multi-walled carbon nanotubes (MWCNTs–COOH) were used as adsorbent for the separation of flavonoids (naringin and rutin) from bitter orange peel. The influence of the parameters such as, pH values, contact time, and desorption conditions was investigated. The samples were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, derivative thermogravimetric, scanning electron microscopy, UV–Vis spectroscopy, and high-performance liquid chromatography. After separation and desorption process, the eluent was injected for chromatography analysis. Under the optimal conditions, experimental results showed that the extraction efficiency of rutin was higher than naringin and other compounds. Moreover, the desorption percentage of flavonoids was calculated 83.6% after four cycles. This research confirmed that this method for separation of flavonoids is simple and less cost. In addition, the separated flavonoids can be used as antioxidant for the future applications.