A magnetic solid-phase extraction (SPE) method based on polystyrene maleic anhydrid/magnetite (Fe3O4) Nano-hybrid as an innovative adsorbent was developed for the separation and pre-concentration of atenolol prior to its determination by spectrofluorometry. This prepared SMA/ Fe3O4Nano-hybrid possesses the magnetic property of Fe3O4 Nano-particles that makes it easily manipulated by an external magnetic field. On the other hand, the surface modification of SMA by APTES leads to selective separation of the target analyte from sample matrices. The structure and morphology of the synthesized adsorbent were characterized using powder X-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The experimental factors affecting the extraction/pre-concentration and determination of the analyte were investigated and optimized. Under the optimized experimental conditions, the calibration graph was linear in the range between 0.10 and 60 ng mL−1, with a correlation coefficient of 0.9989. The limits of detection and enrichment factor for atenolol were 0.32 ng mL−1 and 100 ng mL−1, respectively. The maximum sorption capacity of the adsorbent for atenolol was 96.7 mg g−1. The method was successfully applied to monitoring atenolol in human serum and pharmaceutical waste-waters samples, with recoveries in the range of 96.0%-103.0% for the spiked samples.