Sirolimus (Sir) is widely used in organ transplant patients because it inhibits the mechanistic target of rapamycin (mTOR) activity and suppresses the immune system. In addition, Sir affects translation, biological processes, as well as various other cell signaling pathways. Therefore, it is essential to determine its systemic concentration clinically. However, determining Sir concentration using optical, chromatographic, and mass spectrometry techniques is expensive, time-consuming, and technically challenging. Here, we used an ISFET-based electrochemical sensor, based on the NH2-MIL-53(Fe) sensing layer, to detect Sir. NH2-MIL-53(Fe) to iron and amine active functional groups is quickly immobilized on the surface of ISFET with a ZnO-sensitive layer. It detects Sir using adsorption, redox process, and high electron transfer ability. The proposed sensor detected Sir with LOD and LOQ 33.8 nM and 102.43 nM, respectively. In addition, it had a high sensitivity of 80.109 mu A.nM- 1 and 28.688 mu A.nM- 1, respectively in buffer and urine. Also, this sensor had reproducibility of results with RSD 2.79 % and reusability in seven consecutive days (76.93-100 % recovery). The NH2-MIL-53(Fe)/ZnO@ISFET is proposed as a cheap, miniaturizable, high-sensitivity real-time sensing method.