Organic nanoparticles (O-dots) with a high photoluminescence quantum yield (94%) and long-lived delayed emission (3.1 μs) originating from thermally activated delayed fluorescence (TADF) were developed as glassy state particles using an oil in water emulsion under high pressure (<20 bar). The TADF glassy O-dots exhibit not only good dispersibility and high photostability in water but also good uptake properties into living cells. The glassy O-dots will open new uses as organic emitters in biological applications.