Imipramine hydrochloride (IMIP) is a tricyclic antidepressant utilized in the treatment of depression and chronic pain in some certain cases together with pain medication. The side effects of anxiety, insomnia, crying attacks, personality change and tachycardia are seen in imipramine overdose; therefore, determination of imipramine is an important issue. In this study, a novel potentiometric PVC membrane ion-selective sensor (ISE) was developed for monitoring of IMIP. MIL-53(Al) metal–organic framework was utilized for the first time as an electroactive material in the construction of imipramine-selective PVC membrane sensor. The sensor membrane consisting of 3.0% MIL-53(Al), 64.0% dibutylphthalate (DBP), 32.0% polyvinylchloride (PVC) and 1.0% potassium tetrakis(4-chlorophenyl)borate (KTPClPB) exhibited the most satisfied potentiometric performance characteristics. The sensor displayed a linear response for imipramine hydrochloride in the concentration range of 1.0 × 10−7 M-1.0 × 10−1 M with a slope of 57.7 mV/decade and detection limit of 5.0 × 10−8 M. The operational pH range of the sensor was determined as 3.7–8.5. The sensor showed highly reproducible and stable potentiometric responses with the response time of less than 5 s. The IMIP content of a pharmaceutical used in the treatment of depression was successfully determined with the proposed imipramine-selective sensor. Additionally, the analytical applicability of the sensor in real biological samples was demonstrated by performing imipramine determinations in spiked human blood serum and urine samples.