Herein, Ag-containing carbon dots (Ag-CDs) was synthesized based on folic acid. In a neutral solution, its fluorescence emission owns a structure fixing fluorescent species with the emission maximum at 400 nm and an excitation-wavelength dependent fluorescent species, respectively. By comparing fluorescent emission and excitation spectra, the electronic absorption origins of these fluorescent species were assigned. With the assistance of UV–Vis absorption and XPS, the pH-regulating fluorescence mechanism of Ag-CDs was studied and proposed. A particularly strong fluorescence emitter was observed at pH ~12 with a mixing coordination structure as Ag(CDs-NH2)OH. The as-prepared Ag-CDs might be developed into a fluorescent sensor, especially at extremely basic conditions.