In this paper, for the first time, a combination of hollow fiber based liquid 3-phase microextraction with a potentiometric method was applied as a highly selective and sensitive method of analysis. Desipramine, as a model compound, was extracted from a small volume in the presence of 0.10 mol L−1 NaOH (donor solution) through a thin phase of propyl benzoate inside the pores of a polypropylene hollow fiber and finally into a 10 μL acidic acceptor solution inside the hollow fiber. Two microelectrodes were designed and inserted into the two ends of a hollow fiber inside the acceptor solution. Potentiometric analysis was performed in situ within an extraction time of 15 min; the final stable signal was used for analytical application. Under the optimized conditions, an enrichment factor of 296 was achieved and the relative standard deviation (RSD%) of the method was 4.5%. The calibration curve was obtained in the range of 3.0 × 10−5 to 3.4 × 10−8 mol L−1 with a reasonable linearity (R2 > 0.9849) and a limit of detection (LOD) of 3.1 × 10−8 mol L−1. Finally, the applicability of the proposed method was evaluated by extraction and determination of desipramine in plasma and urine samples without any special pretreatment.