n-Butylidenephthalide (BP) is an active compound in Angelica sinensis (Danggui) and a potential anti-cancer drug. Thus, purification of BP from complex herb extract samples is important. In this study, we develop a facile method for selective purification of BP from complex samples by using iron oxide magnetic nanoparticles (Fe3O4 MNPs) immobilized with riboflavin-5′-phosphate (RFMP) as affinity probes. RFMP is a polyaromatic compound that contains a phosphate, which can readily chelate with the metal center on the surface of Fe3O4 MNPs. The aromatic rings on RFMP can interact with BP (Kd = ∼6.73 × 10-5 M) through π-π stacking. Thus, Fe3O4@RFMP MNPs are fabricated by simply mixing Fe3O4 MNPs with RFMP under microwave heating (power: 540 W) for 3 min. The as-prepared Fe3O4@RFMP MNPs can be used as affinity probes to selectively concentrate BP from an acidic solution; BP is then released from the MNPs by using acetonitrile. The recovery of BP from the Fe3O4@RFMP MNP-BP conjugates is estimated to be ∼100%. This study also demonstrates the feasibility of using the proposed approach to selectively extract BP from complex A. sinensis extract samples.