A simple, rapid and sensitive analytical method for preconcentration and determination of pramipexole in different biological samples has been developed using solvent bar microextraction (SBME) combined with HPLC-UV. The target drugs were extracted from 10 mL of basic aqueous sample solution into an organic extracting solvent located inside the pores of a polypropylene hollow fiber, then back-extracted into an acidified aqueous solution in the lumen of the hollow fiber. In order to obtain high extraction efficiency, the effect of different variables on the extraction efficiency was studied simultaneously using an experimental design. The experimental parameters of SBME were optimized using a Box-Behnken design after a Plackett-Burman screening design. Under the optimized conditions, an enrichment factor up to 96 was achieved and the relative standard deviation of the method was 4.64% (n = 5). The linear range was 0.05-2000 µg/L with a correlation coefficient (r) of 0.987. Finally, the applicability of the proposed method was evaluated by extraction and determination of pramipexole in plasma and urine samples. The results indicated that SBME method has excellent clean-up and high preconcentration factor and can serve as a simple and sensitive method for analysis of pramipexole in biological samples.