Herein, we present a novel strategy based on a "turn-on" persistent luminescence imaging chemical system of graphitic carbon nitride for detecting biothiols in biological fluids. Graphitic carbon nitride (g-C3N4) as persistent luminescence probe is fabricated via a new procedure based on pyrolysis of guanidine hydrochloride under ambient atmospheric conditions. The prepared g-C3N4 nanosheets give intensively long-persistent luminescence that can avoid interference from biological media such as tissue autofluorescence and scattering light. The original persistent luminescence of g-C3N4 turns off due to the adsorption of silver ion (Ag(+)) onto g-C3N4 materials with an electron transfer process. The presence of biothiols induces the onset of persistent luminescence emission by interrupting the quenching interaction, thereby turning on the imaging probe. The approach exhibits high specificity and high sensitivity to biothiols with low detection limit for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) with 6.4, 8.1, and 9.6 nM, respectively. It is also successfully applied for imaging detection of biothiols in human urine, plasma, and cell lysates, demonstrating its great value of practical application in biological systems.