Fentanyl is a potent narcotic drug with the same effects as morphine or heroin, but it’s significantly more potent than these drugs. That means a tiny dose can have a dangerous impact and is also lethal, so it is essential to measure it. In this work, we have developed a new electrochemical sensor to measure this deadly drug utilizing a nanocomposite of multi-walled carbon nanotube, hydroxyapatite, and copper-based metal–organic framework (MWCNT-HA/Cu-H₃BTC). The nanocomposite was first examined by X-ray diffraction, field emission scanning electron, Infrared, and Raman spectroscopic techniques. The glassy carbon electrode (GCE) modified with MWCNT-HA/Cu-H₃BTC was employed to determine fentanyl in aqueous solutions. The highest oxidation current was generated for fentanyl at GCE/MWCNT-HA/Cu-H₃BTC compared to the GCE, GCE/MWCNT, GCE/MWCNT/HA, and GCE/Cu-H₃BTC. The GCE/MWCNT-HA/Cu-H₃BTC showed a linear relationship between the concentration and the oxidation current of fentanyl in the 0.01 to 100 μM with a detection limit of 3 nM. Finally, the fentanyl quantification in blood serum samples was successfully performed. The GCE/MWCNT-HA/Cu-H₃BTC's reproducibility and stability were indeed excellent.