Doxorubicin (DOX) and Dasatinib (DAS) are important chemotherapy medications that are widely used to treat a variety of cancers, but unfortunately, despite the many benefits of DOX, this drug has numerous reported complications. Therefore, the concurrent administration of DAS and DOX can help better treat breast cancer. The current attempt was made to produce Ni−Fe layered double hydroxide (Ni−Fe LDH) to construct a novel selective and sensitive electrochemical DOX sensor. The characteristics of synthesized Ni−Fe LDH was determined by X-ray diffraction, Fourier transform infrared spectrometry, and f ield emission scanning electron microscopy. The sensor fabrication was based on the surface modification of a screen-printed electrode (SPE) with Ni−Fe LDH (Ni−Fe LDH/SPE). The Ni−Fe LDH/SPE possessed an appropriate response and an appreciable electrocatalytic behavior toward the detection of DOX and DAS. There was a significant enhancement in the DOX and DAS electro-oxidation signals on the Ni−Fe LDH/SPE surface compared to that on the bare SPE. The proposed protocol was explored by chronoamperometry, cyclic voltammetry, and differential pulse voltammetry (DPV). The DPV findings clarified a broad linear relationship between DOX concentrations, and the peak height ranged from 0.04 to 585.0 μM with a limit of detection as narrow as 0.01 μM. The practical potency of Ni−Fe LDH/SPE was confirmed by sensing DOX and DAS in biological matrices.