The development of an ultrafast folic acid (FA) sensor (with no incubation time), having a detection limit and detection range in the nanomolar levels is of high importance in the field of biomedical FA sensing. For that, highly luminescent carbon dots (CDs) are synthesized by hydrothermal method with a lower growth duration of ‘1 h’ using the precursors, anhydrous citric acid as the carbon source and ethylenediamine as the nitrogen dopant material. TEM images confirmed a spherical morphology for the prepared CDs with an average size of nearly 2.8 nm. A photoluminescence quenching-based technique is used for FA detection using the prepared CDs. The detection limit is 6 nM, the lowest reported for CDs made from these precursors and the detection range is 0–50 nM. The mode of quenching is found to be static, based on the lifetime measurements and UV–vis spectroscopy. The mechanism of static quenching due to the formation of a ground state complex between the synthesized CDs and FA is confirmed by Raman spectroscopy and dynamic light scattering (DLS) study, and the binding mechanism of CDs and FA is explained in detail using FTIR and Raman spectroscopy for the first time. The fabricated CDs are used as biocompatible probes to image the N2a cells. A quantitative reduction in fluorescence intensity was observed for CD + FA mixtures internalized into the cells with incremental addition of FA. Furthermore, the practicality of the CD probe as an FA sensor is evaluated by testing the amount of FA in spiked plasma samples from albino rats, and the results are validated using a clinical method.