This work has prepared a hyper-crosslinked molecularly imprinted resins (MIRs) with chloromethylated polystyrene resin as a carrier through post-crosslinked Friedel-Crafts and surface imprinting. The prepared MIRs were used for the selective separation and purification of camptothecin (CPT). The prepared MIRs are grafted with a large amount of active group phenol, which have good hydrophilicity and are favorable for application in aqueous environments. The results of Scatchard analysis and contact angle test have proved that MIRs have good adsorption capacity and excellent hydrophilicity. In addition, the results of density functional theory (DFT) analysis show that the possible interaction forces between the template molecule CPT and the functional monomer phenol are the hydrogen bonds and electrostatic attraction. Moreover, MIRs are successfully used as a solid phase extraction (SPE) adsorbent to separate and purify CPT from Camptotheca acuminata fruit. The eluent obtained by SPE is analyzed through HPLC. The detection limit for CPT is 0.23 μg mL⁻¹. The established method preforms high the recovery of CPT (91.2–103.4%) with relative standard deviation less than 4.5%. The reusability of MIRs adsorbent is also investigated, and the adsorption efficiency is still above 95% after being recycled five times. The study provides a feasible and efficient method for selective separate and purify CPT from complicated system.