Herein, we designed a sensitive and selective “Turn-On” fluorescence nanosensor using water-soluble carbonaceous fluorescent nanomaterials (CFNs) functionalized with thiourea (CFNs-Thiourea) for efficient detection of trace concentrations of arsenic (III) in aqueous samples. The CFNs and CFNs-Thiourea were characterized by transmission electron microscopy (TEM), UV–visible spectroscopy (UV-vis) and fourier transformed infrared spectroscopy (FTIR). The emission peak intensity of proposed nanosensor at 425 nm was gradually enhanced on arsenite addition in a wide detection range (3.3–828.5 μg L−1) attributed to the binding of arsenite species with sulfur groups of CFNs-Thiourea. The limit of detection (LOD) was 0.48 μg L−1 being much lower than the World Health Organization (WHO) recommended threshold value of 10 μg L−1. Furthermore, the as-prepared CFNs-Thiourea exhibited a superb selectivity for As (III) compared to various cations and anions, such as; NO3−, NO2−, F−, Ni2+, Fe3+, Cu2+, Ca2+, Mg2+, Zn2+, Fe2+, Hg2+, Pb2+, F−, Cl−, Mn2+, Cr3+, Co2+, Cd2+, Bi3+, Al3+ and As (V) at 100 folds concentration of As (III). The turn on fluorescence nanosensor was successfully exploited for quantification of arsenic in spiked water samples with acceptable efficiencies.