Aniline has been classified as a probable human carcinogen which can cause severe respiratory tract irritation and congestion to humans. Here, we propose using ultra-small (1.8 nm) colloidal nitrogen-doped carbon nano-dots (N-CDs), that is green synthesized at a low temperature of only 100 °C from ultrasonic-processed water-soluble folic acid, as a novel turn-off fluorescent chemo-sensor for ultrasensitive detection of aniline in aqueous medium. It was found that the Stern-Volmer relation describes very well the fluorescence quenching of N-CDs by aniline and confirms a static quenching mechanism between them. Compared to other techniques, our N-CDs based optical detection method, is the simplest and the most ultrasensitive method for aniline liquid detection, reaching an exceptional low detection limit of 3.57 nM (0.332 ppb). The proposed method was successfully applied for determination of aniline in a tap water sample without significant interferences from the matrix.