Objective To prepare methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) nanoparticles loading a water-quenching fluorescent probe and to evaluate its in vitro characteristics and stability. Methods PCL nanoparticles and mPEG-PCL nanoparticles with different mPEG chain lengths (mPEG_5k, mPEG_2k) loading the water-quenching fluorescent probe P2 were prepared by an emulsification/solvent evaporation method using PCL and mPEG-PCL as materials. In vitro characteristics such as morphology, particle size and distribution were evaluated. The P2 probe displayed fluorescent signals when encapsulated in the matrix of the nanoparticles, but quenched immediately when released into water. Based on this properties of P2 probe, the stability of various nanoparticles in different aqueous media was investigated. Results The prepared mPEG-PCL nanoparticles showed a particle size of about 200 nm, narrow size distribution, polydispersity index below 0.06 and near neutral surface potentials with spherical morphology and smooth surfaces. In buffers of different pHs and simulated bio-relevant media, all nanoparticles showed very good stability without significant change in particle size, polydispersity index and fluorescence intensity. Conclusion mPEG-PCL nanoparticles have promising in vitro characteristics and robust stability. It is fast and convenient to monitor the stability of nanoparticles by using the water-quenching probe.