Synthesis of atomically thin tin oxide nanosheets using a nonhydrolytic sol–gel method at ambient temperature is reported in the present work. After 10 min of probe sonication and 7 days of incubation at ambient temperature, ultrathin tin oxide nanosheets (SnO_NS) from 2 nm to 5 μm were formed due to interaction between tin chloride and acetone. The most upsurging property of SnO_NS was the inherent yellow fluorescence (λ_ex= 420 and λ_em= 540 nm) phenomenon rarely reported with respect to tin-based nanosheets. The relative quantum yield of SnO_NS was ∼16%. The origin of fluorescence in SnO_NS is speculated to originate from multiple transitions in oxygen states of Sn, as confirmed by X-ray photoelectron spectroscopy. For biological applications, SnO_NS was phase transferred from acetone to water without compromising the fluorescent properties of SnO_NS. Image cytometry (Nucleo-Counter NC-3000) and laser confocal microscopy were used for comprehending the cellular biocompatibility and internalization of nanoparticles inside the cells (biological imaging). With respect to control (100%), viability and vitality of the cancer cells were found to be 60.4% and 64%, respectively, after 12 h of interaction with SnO_NS.