In the present study, a modified carbon nano-onion and β–cyclodextrin composite was utilized in micro-solid phase extraction of atenolol and metoprolol from human plasma samples. The synthesized sorbent structure and morphology were evaluated. In this method, a proper amount of the sorbent was contacted with the diluted plasma sample under vortexing (to promote the sorbent contact area with the solution). The separated particles by centrifugation were contacted with a proper eluent to desorb the target analytes. After that, the eluent was separated by centrifugation and used during analysis of the analytes by high-performance liquid chromatograph. Several effective extraction parameters such as the sorbent amount, type and elution solvent volume, and extraction time were evaluated and optimized. The validated factors showed that detection and quantification were 0.09 and 0.01 and 0.29 and 0.03 ng mL–1, for metoprolol and atenolol, respectively. The method precision, considered as relative standard deviation, was ≤ 2.5 and ≤ 3.6% for intra- (n = 5) and inter-day (n = 4) precisions, respectively. The extraction recovery values were obtained 84 and 63% for metoprolol and atenolol, respectively. Lastly, the developed method was utilized successfully on the plasma samples of patients treated with metoprolol and atenolol.