This study presents a novel, sensitive and selective molecularly imprinted solid-phase extraction (MISPE)-spectrofluorimetric method for the removal and determination of atenolol from human urine. Molecularly imprinted and non-imprinted polymers were synthesized thermally using a radical chain polymerization technique and used as solid-phase extraction sorbents. Acrylic acid ethylene glycol dimethacrylate, dibenzoyl peroxide and dichloroethane were used as a functional monomer, cross-linker, initiator and porogen, respectively. The calibration curve was in the range of 0.10-2.0 μg/ml for the developed method. Limit of detection and limit of quantification values were 0.032 and 0.099 μg/ml, respectively. Owing to the selectivity of the MISPE technique and the sensitivity of spectrofluorimetry, trace levels of atenolol have been successfully determined from both organic and aqueous media. Relatively high imprinting factor (4.18) and recovery results (74.5-75.3%) were obtained. In addition, intra- and interday precision values were 0.38-1.03% and 0.47-2.05%, respectively, proving the precision of the proposed method. Thus, a selective, sensitive and simple MISPE-spectrofluorimetric method has been developed and applied to the direct determination of atenolol from human urine.