As a chemotherapy drug, 5‐fluorouracil (5‐FU) has been used for colon cancer for decades. Excessive levels of 5‐FU in the human body can lead to notable adverse effects, including severe diarrhea, infection, mouth sores, skin peeling, skin inflammation, and ulcers, which are important and relatively common digestive side effects. In addition, 5‐FU is an analog of uracil and also has similarities to pyrimidines. Therefore, it is not easy to separate them. This research presented a sensor capable of detecting drugs in minimal amounts. An alginate‐derived carbon dot (CD) was synthesized by unique optical properties that obey an on–off fluorescence mechanism for 5‐FU sensing. Introducing copper (Cu(I)) to CDs results in fluorescence quenching through electron transfer. However, when 5‐FU is added to the system as an oxidizing agent, a redox reaction occurs on the surface of the CDs, which leads to the restoration of fluorescence as Cu(I) is altered to Cu(II). Experimental results showed a strong linear correlation (R2 = 0.99) in the concentration range of 1.00–45.00 nM, with the following linear regression, and revealed the relative standard deviation (RSD%) and detection limit of 2.57%, and 1.00 nM, respectively. These results validated the excellent detection capability of the proposed method even at low concentrations of 5‐FU and in the presence of other drugs and interfering substances. Also, the recovery of 5‐FU (varies from 100.46% to 113.7%, with RSD equal to 1.89–3.63) in serum samples indicates the absence of matrix interference in the determination of 5‐FU. In summary, this novel approach to developing a cost‐effective and sensitive sensor holds great potential for future applications in healthcare and related fields.