Carbon dots as fluorescent probes exhibit brilliant sensitivity and selectivity in copper ion detection, which greatly depends on their microstructure. Herein, a mild oxidation method was adopted to tailor the organic macromolecules of coal and its derivatives for preparing coal-based carbon dots. Such coal-based carbon dots comprise a carbonaceous center dominated by sp2 microcrystalline carbon, surface defects formed by sp3 amorphous carbon, and oxygen/nitrogen-containing functional groups. The coal-based humic acid-based carbon dots (HA-CDs) exhibit high dispersity (∼7.6 nm), high sp2 microcrystalline carbon (61.73 %), abundant carboxyl groups (2.09 %), hydroxy groups (8.93 %), and pyridinic nitrogen (1.06 %). Due to their unique microstructure, the HA-CDs in aqueous solutions exhibit prominent fluorescence stability under different pH conditions and show superior selectivity and sensitivity to Cu2+ even in the existence of other metal ions. Within the concentration range of 0–40 µmol/L, the fluorescence intensity of HA-CDs linearly correlates with the concentration of Cu2+. The limit of detection is 0.014 µmol/L, allowing for tracing the Cu2+ concentration in flowing water. This work affords a novel idea for the microstructure design of high-sensitivity carbon dots for Cu2+ detection in the water environment.