The present study combines UV-Vis spectrophotometry and dispersive liquid-liquid microextraction (DLLME) for the preconcentration and determination of trace level clidinium bromide (Clid) in pharmaceutical preparation and real samples. The method is based on ion-pair formation between Clid and bromocresol green in aqueous solution using citrate buffer (pH = 3). The colored product was first extracted using a mixture of 800 µL acetonitrile and 300 µL chloroform solvents. Then, a spectrophotometric measurement of sediment phase was performed at λ = 420 nm. The important parameters affecting the efficiency of DLLME were optimized. Under the optimum conditions, the calibration graphs of standard -1 (Std.), drug, urine and serum were ranged 0.005 - 0.16 µg mL . The limits of detection, quantification, and Sandell's sensitivity were calculated. Good recoveries of Clid Std., drug, urine and serum at 0.005, 0.01, -1 0.1 and 0.16 µg mL ranged 93.77 - 101.0%. Enrichment factor was calculated for Std., drug, urine and serum. The method was applied successfully to determine Clid in pharmaceutical preparation and real samples.