A novel one-step method for the synthesis of bright, multicolor fluorescent sulphur doped carbon dots (CNDs) has been developed by using simple microwave assisted pyrolysis of citric acid and sodium thiosulphate. The synthesized CNDs showed dual mode naked eye colorimetric ultrasensitive sensing capability both for arsenic [As (III)] and glutathione (GSH) with high selectivity. Using fluorometric assay, the detection limit (DL) for As (III) was found to be as low as 32 pM. The selectivity data show that the newly developed CNDs is very specific for As (III) even with interference by high concentrations of other metal ions. The CNDs were also able to detect GSH very selectively over other biothiols like cysteine (Cys) and homo-cysteine (H-cys) with a DL of 43 nM, even in blood plasma. The fast kinetic data suggests that the present CNDs assay could be used onsite As (III) detection. The CNDs, further, showed its potential application in high resolution bioimaging of bacterial nucleoid segregation.