This study aims to engineer a new type of ultrahigh quantum yield carbon dots (CDs) from methotrexate (MTX-CDs) with self-targeting, imaging, and therapeutic effects on MDA-MB 231 breast cancer cells. CDs were synthesized via a straightforward thermal method using a methotrexate (MTX) drug source. The physicochemical characteristics of the prepared MTX-CDs were studied using Fourier transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray powder diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). TEM and DLS revealed which MTX-CDs have homogeneous spherical morphology with a smaller average size of 5.4 ± 2.2 nm, polydispersity index (PDI) of 0.533, and positive surface charge of around +3.93 mV. Results of FT-IR spectroscopy and high-resolution XPS indicated the presence of residues of MTX on CDs. Therefore, the synthesized MTX-CDs could be targeted and be taken up by FR-positive cell lines without the aid of additional targeting molecules. In vitro epifluorescence images demonstrated high-contrast cytoplasm biodistribution of MTX-CDs after 2 h of treatment. A much stronger fluorescent signal was detected in MDA-MB 231 compared to MCF 7, indicating their ability to precisely target FR. The highest cytotoxic and apoptotic effects were observed in MTX-CDs compared to free MTX obtained by the MTT assay, cell cycle arrest, and annexin V-FITC apoptosis techniques. Results revealed that the novel engineered MTX-CDs were capable of inducing apoptosis (70.2% apoptosis) at a lower concentration (3.2 μM) compared to free MTX, which was proved by annexin V and cell cycle. This work highlights the potential application of CDs for constructing an intelligent nanomedicine with integration of diagnostic, targeting, and therapeutic functions.