We here designed and prepared a multi-responsive microgel (hPEA-mGel) fabricated through the co-assembly of hyperbranched poly(ether amine) and pentaerythritol tetra(3-mercaptopropionate) (PTMP) in aqueous solution, followed by thiol–ene photo-click crosslinking. The obtained hPEA-mGel is uniform-sized particles with 250 nm diameter and low polydispersity index (PDI = 0.15), and exhibited sharp responses to temperature and pH. The interaction between the host hPEA-mGel and a series of fluorescein dyes was investigated. hPEA-mGel exhibited strong adsorption to ETB, RB, EB, DBF and TCF, but very low affinity to FR and Cal. The big difference in the interaction between hPEA-mGel and different fluorescein dyes is indicative of the selective adsorption of hPEA-mGel to fluorescein dyes, which resulted from hydrophobic not electrostatic interactions. Based on the selective adsorption of fluorescein dyes by hPEA-mGel, we demonstrated a simple approach to separate a mixture of fluorescein dyes in aqueous solution.