We here reported that hyperbranched poly(ether amine) (hPEA) and poly(vinyl alcohol) (PVA) interpenetrating network (hPEA/PVA-IPN) can be used for the selective adsorption and separation of guest homologues. A series of hyperbranched poly(ether amine) and poly(vinyl alcohol) interpenetrating networks (hPEA/PVA-IPNs) were fabricated by introducing poly(vinyl alcohol) chains into network of hyperbranched poly(ether amine), in which two independent networks of hyperbranched poly(ether amine) and poly(vinyl alcohol) were cross-linked through photodimerization of coumarin groups of hyperbranched poly(ether amine) and aldol condensation reaction between hydroxyl groups of poly(vinyl alcohol) and glutaraldehyde, respectively. The mechanical strength of interpenetrating networks can be enhanced by the introduction of poly(vinyl alcohol), and the tensile strength of interpenetrating networks increased with tens of times in compared with the pure hyperbranched poly(ether amine) network. The adsorption behavior of seven fluorescein dyes sharing with the same backbone and charge state onto hyperbranched poly(ether amine) and poly(vinyl alcohol) interpenetrating networks was then investigated in detail. Regardless of their charge states, these interpenetrating networks exhibited the quick adsorption to Rose Bengal (RB), Erythrosin B (ETB), Eosin B (EB), 4',5'-dibromofluorescein (DBF), and 4,5,6,7-tetrachlorofluorescein (TCF), with high adsorption capacity (Qeq) and very low adsorption of Calcein (Cal) and fluorescein (FR). The adsorption process was found to follow the pseudo-second-order kinetics, and the introduction of poly(vinyl alcohol) has no obvious effect on the adsorption behavior in this study. The big difference in the adsorption is indicative of the selective adsorption of hyperbranched poly(ether amine) and poly(vinyl alcohol) interpenetrating networks to fluorescein dyes. Based on the unique selective adsorption, the separation of several mixtures of fluorescein dyes such as RB/Cal, RB/FR, ETB/FR, and ETB/Cal was achieved by using hPEA/PVA-IPN as adsorbent.