To establish a new method for detecting crystal violet (CV), a harmful dye, herein, a genre of novel biomass carbon dots (CDs) was synthesized via a microwave method and employed as a fluorescent probe, in which water spinach and polyethylene glycol (PEG) performed as raw materials. Based on the inner filter effect (IFE) between the luminescent CDs and CV, the blue emission of this probe at 430 nm could be quenched by CV. Hence, a new strategy was proposed to selectively determine CV in aquaculture ambient. Moreover, under the optimal experiment conditions, this method showed a good linearity between the concentration of CV (c) and fluorescence quenching rate (ΔF/F0) in the concentration range of 4–200 μmol/L with the corresponding correlation coefficient (r) and the detection limit of 0.997 and 710 nmol/L, respectively. With advantages of environmental protectivity, sensitivity, affordability, and user‐friendliness, the facilely fabricated CDs could be successfully applied in detecting CV in aquaculture samples, providing a technical foundation for monitoring the pollution of CV and ensuring the quality and safety of aquatic products.