Abstract The novel photoresponsive molecularly imprinted polymers conjugated hyperbranched polymers based on functionalized magnetic nanoparticles (PMIP@HBPM) were developed for selective recognition and intelligent separation of azathioprine from the complex samples. The PMIP was prepared using an azobenzene-containing monomer as the photosensitive monomer by surface imprinting technology and the stimuli responsive mechanism. The structure and physical property of newly synthesized materials have been characterized by different spectroscopic and nanostructural techniques. The release and uptake of azathioprine from PMIP@HBPM is photo-regulated by alternate irradiation at 365 nm and 440 nm, indicating that template molecule can be easily bound in the recognition sites and released back into the solution. Subsequently, the PMIP@HBPM was also successfully applied to photo-controlled extraction of azathioprine in biological and pharmaceutical samples with good recovery, high accuracy and precision, confirming great applicability for trace determination of a specific analytes from complex samples. The present work mainly provided the useful insights and deep analysis about the feasibility of using hyperbranched polymers-magnetic nanoparticles and stimuli responsive imprinted materials in smart separation system, which displays great potential to create a sustainable environment.