Waste utilization is not only a way to protect the environment and realize green chemistry, but also a means to create novel materials. In this study, based on waste grape seeds as the biowaste-derived carbon dots (G-BCDs), a straightforward one-pot green method was employed for the rapid detection of folic acid (FA). Owing to the internal filter effect and the static mixing quenching mechanism, the sensing principle of G-BCDs was effectively quenched by FA. The results showed fluorescence at an emission wavelength of 415 nm upon excitation at 330 nm with a quantum yield of 1.5%. Particularly, the FA sensing assay obtained a broad linear range of 2–220 μM and the limit of detection was 0.48 μM. In addition, the fluorescence probe was successfully utilized for detecting FA in tablets, blood, and urine samples, yielding desirable results, which indicated promising applications in the fields of biological and pharmaceutical analysis.