A hollow-fiber liquid-phase microextraction (HF-LPME), followed by high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the trace determination of carvedilol (β-blocker) in biological fluids, has been described. The separation was achieved using Inertsil ODS-3 C18 (250 mm � 4.6 mm, 3 μm) column with a mobile phase composition of 10 mM phosphate buffer (pH 4.0)-acetonitrile (50:50, v/v) at a flow rate of 1.0 mL/min, under isocratic elution. Several parameters (i.e., type of organic solvent, donor phase pH, concentration of acceptor phase (AP), stirring rate, extraction time, and salt addition) that affect the extraction efficiency were investigated. The optimum HF-LPME conditions were as follows: Dihexyl ether as an organic solvent; donor phase pH, 10.7; 0.1 M HCl (AP); 1100-rpm stirring rate; 60-min extraction time; and no salt addition. These parameters have been confirmed using design of experiments. Under these conditions, an enrichment factor of 273-fold was achieved. Good linearity and correlation coefficient were obtained over the range 5-1000 ng/mL (r2 = 0.9994). Limits of detection and quantitation were 1.2 and 3.7 ng/mL, respectively. The relative standard deviation at 3 different concentration levels (5, 500, and 1000 ng/mL) were less than 13.2. Recoveries for spiked urine and plasma were in the range 80.7-114. The proposed method is simple, sensitive, and suitable for the determination of carvedilol in biological fluids. ©2019 The Author(s).